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Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1beta and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death.

Reduced saturated fat (SFA) consumption is recommended to reduce coronary heart disease (CHD), but there is an absence of strong supporting evidence from randomized controlled trials (RCTs) of clinical CHD events and few guidelines focus on any specific replacement nutrient. Additionally, some public health groups recommend lowering or limiting polyunsaturated fat (PUFA) consumption, a major potential replacement for SFA. We systematically investigated and quantified the effects of increased PUFA consumption, as a replacement for SFA, on CHD endpoints in RCTs. RCTs were identified by systematic searches of multiple online databases through June 2009, grey literature sources, hand-searching related articles and citations, and direct contacts with experts to identify potentially unpublished trials. Studies were included if they randomized participants to increased PUFA for at least 1 year without major concomitant interventions, had an appropriate control group, and reported incidence of CHD (myocardial infarction and/or cardiac death). Inclusions/exclusions were adjudicated and data were extracted independently and in duplicate by two investigators and included population characteristics, control and intervention diets, follow-up duration, types of events, risk ratios, and SEs. Pooled effects were calculated using inverse-variance-weighted random effects meta-analysis. From 346 identified abstracts, eight trials met inclusion criteria, totaling 13,614 participants with 1,042 CHD events. Average weighted PUFA consumption was 14.9% energy (range 8.0%-20.7%) in intervention groups versus 5.0% energy (range 4.0%-6.4%) in controls. The overall pooled risk reduction was 19% (RR = 0.81, 95% confidence interval [CI] 0.70-0.95, p = 0.008), corresponding to 10% reduced CHD risk (RR = 0.90, 95% CI = 0.83-0.97) for each 5% energy of increased PUFA, without evidence for statistical heterogeneity (Q-statistic p = 0.13; I(2) = 37%). Meta-regression identified study duration as an independent determinant of risk reduction (p = 0.017), with studies of longer duration showing greater benefits. These findings provide evidence that consuming PUFA in place of SFA reduces CHD events in RCTs. This suggests that rather than trying to lower PUFA consumption, a shift toward greater population PUFA consumption in place of SFA would significantly reduce rates of CHD. Please see later in the article for the Editors' Summary.

Coronary heart disease (CHD) is the leading cause of mortality in African Americans. To identify common genetic polymorphisms associated with CHD and its risk factors (LDL- and HDL-cholesterol (LDL-C and HDL-C), hypertension, smoking, and type-2 diabetes) in individuals of African ancestry, we performed a genome-wide association study (GWAS) in 8,090 African Americans from five population-based cohorts. We replicated 17 loci previously associated with CHD or its risk factors in Caucasians. For five of these regions (CHD: CDKN2A/CDKN2B; HDL-C: FADS1-3, PLTP, LPL, and ABCA1), we could leverage the distinct linkage disequilibrium (LD) patterns in African Americans to identify DNA polymorphisms more strongly associated with the phenotypes than the previously reported index SNPs found in Caucasian populations. We also developed a new approach for association testing in admixed populations that uses allelic and local ancestry variation. Using this method, we discovered several loci that would have been missed using the basic allelic and global ancestry information only. Our conclusions suggest that no major loci uniquely explain the high prevalence of CHD in African Americans. Our project has developed resources and methods that address both admixture- and SNP-association to maximize power for genetic discovery in even larger African-American consortia.

Single nucleotide polymorphisms (SNPs) on chromosome 9p21 are associated with coronary artery disease, diabetes, and multiple cancers. Risk SNPs are mainly non-coding, suggesting that they influence expression and may act in cis. We examined the association between 56 SNPs in this region and peripheral blood expression of the three nearest genes CDKN2A, CDKN2B, and ANRIL using total and allelic expression in two populations of healthy volunteers: 177 British Caucasians and 310 mixed-ancestry South Africans. Total expression of the three genes was correlated (P<0.05), suggesting that they are co-regulated. SNP associations mapped by allelic and total expression were similar (r = 0.97, P = 4.8x10(-99)), but the power to detect effects was greater for allelic expression. The proportion of expression variance attributable to cis-acting effects was 8% for CDKN2A, 5% for CDKN2B, and 20% for ANRIL. SNP associations were similar in the two populations (r = 0.94, P = 10(-72)). Multiple SNPs were independently associated with expression of each gene (P<0.05 after correction for multiple testing), suggesting that several sites may modulate disease susceptibility. Individual SNPs correlated with changes in expression up to 1.4-fold for CDKN2A, 1.3-fold for CDKN2B, and 2-fold for ANRIL. Risk SNPs for coronary disease, stroke, diabetes, melanoma, and glioma were all associated with allelic expression of ANRIL (all P<0.05 after correction for multiple testing), while association with the other two genes was only detectable for some risk SNPs. SNPs had an inverse effect on ANRIL and CDKN2B expression, supporting a role of antisense transcription in CDKN2B regulation. Our study suggests that modulation of ANRIL expression mediates susceptibility to several important human diseases.

The rapidly evolving field of metabolomics aims at a comprehensive measurement of ideally all endogenous metabolites in a cell or body fluid. It thereby provides a functional readout of the physiological state of the human body. Genetic variants that associate with changes in the homeostasis of key lipids, carbohydrates, or amino acids are not only expected to display much larger effect sizes due to their direct involvement in metabolite conversion modification, but should also provide access to the biochemical context of such variations, in particular when enzyme coding genes are concerned. To test this hypothesis, we conducted what is, to the best of our knowledge, the first GWA study with metabolomics based on the quantitative measurement of 363 metabolites in serum of 284 male participants of the KORA study. We found associations of frequent single nucleotide polymorphisms (SNPs) with considerable differences in the metabolic homeostasis of the human body, explaining up to 12% of the observed variance. Using ratios of certain metabolite concentrations as a proxy for enzymatic activity, up to 28% of the variance can be explained (p-values 10(-16) to 10(-21)). We identified four genetic variants in genes coding for enzymes (FADS1, LIPC, SCAD, MCAD) where the corresponding metabolic phenotype (metabotype) clearly matches the biochemical pathways in which these enzymes are active. Our results suggest that common genetic polymorphisms induce major differentiations in the metabolic make-up of the human population. This may lead to a novel approach to personalized health care based on a combination of genotyping and metabolic characterization. These genetically determined metabotypes may subscribe the risk for a certain medical phenotype, the response to a given drug treatment, or the reaction to a nutritional intervention or environmental challenge.

Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined. Using bone marrow-derived mast cells from wild-type, Tnf(-/-), Ifng(-/-), Il6(-/-) mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC. Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases.

Leukocyte telomere length, an emerging marker of biological age, has been shown to predict cardiovascular morbidity and mortality. However, the natural history of telomere length in patients with coronary artery disease has not been studied. We sought to investigate the longitudinal trajectory of telomere length, and to identify the independent predictors of telomere shortening, in persons with coronary artery disease. In a prospective cohort study of 608 individuals with stable coronary artery disease, we measured leukocyte telomere length at baseline, and again after five years of follow-up. We used multivariable linear and logistic regression models to identify the independent predictors of leukocyte telomere trajectory. Baseline and follow-up telomere lengths were normally distributed. Mean telomere length decreased by 42 base pairs per year (p<0.001). Three distinct telomere trajectories were observed: shortening in 45%, maintenance in 32%, and lengthening in 23% of participants. The most powerful predictor of telomere shortening was baseline telomere length (OR per SD increase = 7.6; 95% CI 5.5, 10.6). Other independent predictors of telomere shortening were age (OR per 10 years = 1.6; 95% CI 1.3, 2.1), male sex (OR = 2.4; 95% CI 1.3, 4.7), and waist-to-hip ratio (OR per 0.1 increase = 1.4; 95% CI 1.0, 2.0). Leukocyte telomere length may increase as well as decrease in persons with coronary artery disease. Telomere length trajectory is powerfully influenced by baseline telomere length, possibly suggesting negative feedback regulation. Age, male sex, and abdominal obesity independently predict telomere shortening. The mechanisms and reversibility of telomeric aging in cardiovascular disease deserve further study.

Circulating endothelial progenitor cells (EPC), involved in endothelial regeneration, neovascularisation, and determination of prognosis in cardiovascular disease can be characterised with functional assays or using immunofluorescence and flow cytometry. Combinations of markers, including CD34+KDR+ or CD133+KDR+, are used. This approach, however may not consider all characteristics of EPC. The lack of a standardised protocol with regards to reagents and gating strategies may account for the widespread inter-laboratory variations in quantification of EPC. We, therefore developed a novel protocol adapted from the standardised so-called ISHAGE protocol for enumeration of haematopoietic stem cells to enable comparison of clinical and laboratory data. In 25 control subjects, 65 patients with coronary artery disease (CAD; 40 stable CAD, 25 acute coronary syndrome/acute myocardial infarction (ACS)), EPC were quantified using the following approach: Whole blood was incubated with CD45, KDR, and CD34. The ISHAGE sequential strategy was used, and finally, CD45(dim)CD34(+) cells were quantified for KDR. A minimum of 100 CD34(+) events were collected. For comparison, CD45(+)CD34(+) and CD45(-)CD34(+) were analysed simultaneously. The number of CD45(dim)CD34(+)KDR(+) cells only were significantly higher in healthy controls compared to patients with CAD or ACS (p = 0.005 each, p<0.001 for trend). An inverse correlation of CD45(dim)CD34(+)KDR(+) with disease activity (r = -0.475, p<0.001) was confirmed. Only CD45(dim)CD34(+)KDR(+) correlated inversely with the number of diseased coronaries (r = -0.344; p<0.005). In a second study, a 4-week de-novo treatment of atorvastatin in stable CAD evoked an increase only of CD45(dim)CD34(+)KDR(+) EPC (p<0.05). CD45(+)CD34(+)KDR(+) and CD45(-)CD34(+)KDR(+) were indifferent between the three groups. Our newly established protocol adopted from the standardised ISHAGE protocol achieved higher accuracy in EPC enumeration confirming previous findings with respect to the correlation of EPC with disease activity and the increase of EPC during statin therapy. The data of this study show the CD45(dim) fraction to harbour EPC.

Systemic and local inflammation plays a prominent role in the pathogenesis of atherosclerotic coronary artery disease, but the relationship of whole blood gene expression changes with coronary disease remains unclear. We have investigated whether gene expression patterns in peripheral blood correlate with the severity of coronary disease and whether these patterns correlate with the extent of atherosclerosis in the vascular wall. Patients were selected according to their coronary artery disease index (CADi), a validated angiographical measure of the extent of coronary atherosclerosis that correlates with outcome. RNA was extracted from blood of 120 patients with at least a stenosis greater than 50% (CADi > or = 23) and from 121 controls without evidence of coronary stenosis (CADi = 0). 160 individual genes were found to correlate with CADi (rho > 0.2, P<0.003). Prominent differential expression was observed especially in genes involved in cell growth, apoptosis and inflammation. Using these 160 genes, a partial least squares multivariate regression model resulted in a highly predictive model (r(2) = 0.776, P<0.0001). The expression pattern of these 160 genes in aortic tissue also predicted the severity of atherosclerosis in human aortas, showing that peripheral blood gene expression associated with coronary atherosclerosis mirrors gene expression changes in atherosclerotic arteries. In conclusion, the simultaneous expression pattern of 160 genes in whole blood correlates with the severity of coronary artery disease and mirrors expression changes in the atherosclerotic vascular wall.

Genome-wide association studies (GWAS) have linked common single nucleotide polymorphisms (SNPs) on chromosome 9p21 near the INK4/ARF (CDKN2A/B) tumor suppressor locus with risk of atherosclerotic diseases and type 2 diabetes mellitus. To explore the mechanism of this association, we investigated whether expression of proximate transcripts (p16(INK4a), p15(INK4b), ARF, ANRIL and MTAP) correlate with genotype of representative 9p21 SNPs. We analyzed expression of 9p21 transcripts in purified peripheral blood T-cells (PBTL) from 170 healthy donors. Samples were genotyped for six selected disease-related SNPs spanning the INK4/ARF locus. Correlations among these variables were determined by univariate and multivariate analysis. Significantly reduced expression of all INK4/ARF transcripts (p15(INK4b), p16(INK4a), ARF and ANRIL) was found in PBTL of individuals harboring a common SNP (rs10757278) associated with increased risk of coronary artery disease, stroke and aortic aneurysm. Expression of MTAP was not influenced by rs10757278 genotype. No association of any these transcripts was noted with five other tested 9p21 SNPs. Genotypes of rs10757278 linked to increased risk of atherosclerotic diseases are also associated with decreased expression in PBTL of the INK4/ARF locus, which encodes three related anti-proliferative transcripts of known importance in tumor suppression and aging.