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Background Protease inhibitors (PIs) are associated with hypertriglyceridemia and atherogenic dyslipidemia. Identifying HIV-1-infected individuals who are at increased risk of PI-related dyslipidemia will facilitate therapeutic choices that maintain viral suppression while reducing risk of atherosclerotic diseases. Apolipoprotein C-III (apoC-III) gene variants, which vary by race/ethnicity, have been associated with a lipid profile that resembles PI-induced dyslipidemia. However, the association of race/ethnicity, or candidate gene effects across race/ethnicity, with plasma lipid levels in HIV-1-infected individuals, has not been reported. Methods and Findings A cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and PI exposure on plasma lipids was performed in AIDS Clinical Trial Group studies (n = 626). Race/ethnicity was a highly significant predictor of plasma lipids in fully adjusted models. Furthermore, in stratified analyses, the effect of PI exposure appeared to differ across race/ethnicity. Black/non-Hispanic, compared with White/non-Hispanics and Hispanics, had lower plasma triglyceride (TG) levels overall, but the greatest increase in TG levels when exposed to PIs. In Hispanics, current PI antiretroviral therapy (ART) exposure was associated with a significantly smaller increase in TGs among patients with variant alleles at apoC-III-482, −455, and Intron 1, or at a composite apoC-III genotype, compared with patients with the wild-type genotypes. Conclusions In the first pharmacogenetic study of its kind in HIV-1 disease, we found race/ethnic-specific differences in plasma lipid levels on ART, as well as differences in the influence of the apoC-III gene on the development of PI-related hypertriglyceridemia. Given the multi-ethnic distribution of HIV-1 infection, our findings underscore the need for future studies of metabolic and cardiovascular complications of ART that specifically account for racial/ethnic heterogeneity, particularly when assessing candidate gene effects.

Background Endothelial lipase (EL), a new member of the lipase family, has been shown to modulate high-density lipoprotein (HDL-C) metabolism and atherosclerosis in mouse models. We hypothesized that EL concentrations would be associated with decreased HDL-C and increased atherosclerosis in humans. Methods and Findings Healthy individuals with a family history of premature coronary heart disease (n = 858) were recruited as part of the Study of the Inherited Risk of Atherosclerosis. Blood was drawn in the fasting state before and, in a subgroup (n = 510), after administration of a single dose of intravenous heparin. Plasma lipids were measured enzymatically, lipoprotein subclasses were assessed by nuclear magnetic resonance, and coronary artery calcification (CAC) was quantified by electron beam computed tomography. Plasma EL mass was measured using a newly developed enzyme-linked immunosorbent assay. Median EL mass in pre-heparin plasma was 442 (interquartile range = 324–617) ng/ml. Median post-heparin mass was approximately 3-fold higher, 1,313 (888–1,927) ng/ml. The correlation between pre-heparin EL mass and post-heparin EL mass was 0.46 (p < 0.001). EL mass concentrations in both pre- and post-heparin plasma significantly correlated with all NCEP ATPIII-defined metabolic syndrome factors: waist circumference (r = 0.28 and 0.22, respectively, p < 0.001 for each), blood pressure (r = 0.18 and 0.24, p < 0.001 for each), triglycerides (r = 0.22, p < 0.001; and 0.13, p = 0.004), HDL cholesterol (r = –0.11, p = 0.002; and –0.18, p < 0.001), and fasting glucose (r = 0.11 and 0.16, p = 0.001 for both). EL mass in both routine (odds ratio [OR] = 1.67, p = 0.01) and post-heparin (OR = 2.42, p = 0.003) plasma was associated with CAC as determined by ordinal regression after adjustment for age, gender, waist circumference, vasoactive medications, hormone replacement therapy (women), and established cardiovascular risk factors. Conclusions We report, to our knowledge for the first time, that human plasma EL concentrations, in both post-heparin and routine pre-heparin plasma, are significantly associated with metabolic syndrome features and with subclinical atherosclerosis. EL may be a pro-atherogenic factor in humans, especially in overweight individuals and those with metabolic syndrome., Studies in rodents had suggested that endothelial lipase might play a role in the development of atherosclerosis. This study finds an association between EL levels and early-stage atherosclerosis in humans.

BACKGROUND:CKD, an independent risk factor for CV disease, increases mortality in T2DM. Treating modifiable CV risk factors decreases mortality in diabetics with microalbuminuria, but the role of early CV prevention in diabetics with mild CKD by GFR criteria alone remains unclear. The purpose of this study was to probe whether T2DM patients with mild GFR impairment have atherogenic lipid profiles compared to diabetic counterparts with normal renal function. METHODS:In the Penn Diabetes Heart Study (PDHS), a single-center observational cohort of T2DM patients without clinical CVD, cross-sectional analyses were performed for directly measured lipid fractions in 1852 subjects with eGFR>60 mL/min/1.73 m² determined by the CKD-EPI equation (n = 1852). Unadjusted and multivariable analyses of eGFR association with log-transformed lipid parameters in incremental linear and logistic regression models (with eGFR 90 mL/min/1.73 m² as a cut-point) were performed. RESULTS:Mild GFR impairment (eGFR 60-90 mL/min/1.73 m², median urinary ACR 5.25 mg/g) was associated with higher log-transformed Lp(a) values (OR 1.17, p = 0.005) and with clinically atherogenic Lp(a) levels above 30 mg/dL (OR 1.35, p = 0.013) even after full adjustment for demographics, medications, metabolic parameters, and albuminuria. Logistic regression demonstrated a trend towards significance between worse kidney function and apoB (p = 0.17) as well as apoC-III (p = 0.067) in the fully adjusted model. CONCLUSIONS:Elevated Lp(a) levels have a robust association with mild GFR impairment in type 2 diabetics independent of race, insulin resistance, and albuminuria.

Background— Liver X receptors (LXRs) are ligand-activated transcription factors involved in the control of lipid metabolism and inflammation. Synthetic LXR agonists have been shown to inhibit the progression of atherosclerosis in mice, but the mechanism is uncertain. LXR agonism upregulates the genes encoding ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1) in macrophages, thus promoting efflux of cholesterol; it also upregulates liver and intestinal ABCG5 and ABCG8, helping to promote biliary and fecal excretion of cholesterol. Thus, LXR agonism may inhibit atherosclerosis through promotion of reverse cholesterol transport (RCT) in vivo, but this has not been proven. We previously described an in vivo method to trace the movement of cholesterol from 3 H-cholesterol–labeled J774 macrophages into plasma, into liver, and ultimately into the bile and feces as free cholesterol or bile acids. In the present study we used this approach to test the hypothesis that administration of the synthetic LXR agonist GW3965 would increase the rate of macrophage RCT in vivo. Methods and Results— Three different mouse models—wild-type C57BL/6 mice, LDLR/apobec-1 double knockout mice, and human apolipoprotein (apo)B/cholesteryl ester transfer protein (CETP) double transgenic mice—were treated with either vehicle or GW3965. Mice were injected intraperitoneally with 3 H-cholesterol–labeled and cholesterol-loaded macrophages and monitored for the appearance of 3 H-tracer in plasma, liver, and feces. Administration of GW3965 significantly increased the levels of 3 H-tracer in plasma and feces in all 3 mouse models. Conclusions— These results demonstrate that administration of the LXR agonist GW3965 increases the rate of RCT from macrophages to feces in vivo.

Objective— The purpose of this research was to evaluate the short-term effects of pioglitazone (PIO) on high-density lipoprotein cholesterol (HDL-C) and other metabolic parameters in nondiabetic patients with metabolic syndrome (MetSyn). Methods and Results— Sixty nondiabetic adults with low HDL-C and MetSyn were randomized to PIO or matching placebo for 12 weeks. PIO increased HDL-C by 15% and 14% at 6 and 12 weeks, respectively, compared with placebo ( P r =0.34; P =0.01) but not to changes in insulin resistance. PIO did not affect serum triglycerides or low-density lipoprotein (LDL) cholesterol concentrations but reduced the number of small LDL particles by 18% ( P P P =0.02) while increasing mean serum levels of adiponectin by 111% ( P Conclusions— In nondiabetic patients with low HDL-C and MetSyn, PIO significantly raised HDL-C and favorably affected lipoprotein particle size, markers of inflammation, and adipokines without changes in triglycerides, LDL-C, or weight. These results suggest that PIO has direct effects on HDL, which may contribute to its antiatherogenic effects.

We have previously reported suggestive linkage of type 2 diabetes mellitus to chromosome 10q. We genotyped 228 microsatellite markers in Icelandic individuals with type 2 diabetes and controls throughout a 10.5-Mb interval on 10q. A microsatellite, DG10S478, within intron 3 of the transcription factor 7-like 2 gene (TCF7L2; formerly TCF4) was associated with type 2 diabetes (P = 2.1 x 10(-9)). This was replicated in a Danish cohort (P = 4.8 x 10(-3)) and in a US cohort (P = 3.3 x 10(-9)). Compared with non-carriers, heterozygous and homozygous carriers of the at-risk alleles (38% and 7% of the population, respectively) have relative risks of 1.45 and 2.41. This corresponds to a population attributable risk of 21%. The TCF7L2 gene product is a high mobility group box-containing transcription factor previously implicated in blood glucose homeostasis. It is thought to act through regulation of proglucagon gene expression in enteroendocrine cells via the Wnt signaling pathway.

Lipoprotein lipase, HL, and endothelial lipase (EL) are proteoglycan-bound enzymes that regulate plasma lipoprotein levels through coordinated triglyceride (TG) lipase and phospholipase activity. We hypothesized that single nucleotide polymorphisms (SNPs) in lipase genes would have higher order impact on plasma lipoproteins beyond the influence of individual SNPs. In a sample of asymptomatic Caucasian subjects (n = 738), we used a two-stage approach, first identifying groups of subjects with similar multilocus lipase genotypes and then characterizing the relationships between genotype groups and plasma lipids. Using complementary methods, including a permutation test procedure and a mixed-effects modeling approach, we found a higher order interaction between four SNPs in three lipase genes (EL 2,237 3′ untranslated region, EL Thr111Ile, HL −514C/T, and LPL Hind III) and plasma TG levels. Subjects who were heterozygous for all four lipase SNPs had significantly higher plasma TG levels beyond the effect of individual lipase SNPs and environmental factors, even after correcting for multiple comparisons. In conclusion, lipase genes had synergistic association with plasma TG beyond individual gene effects. Higher order multilocus genotype contributions to dyslipidemia and atherosclerotic cardiovascular disease need to be considered a priori because they may have an important effect even in the absence of significant main effects of the individual genes.

Background Protease inhibitors (PIs) are associated with hypertriglyceridemia and atherogenic dyslipidemia. Identifying HIV-1-infected individuals who are at increased risk of PI-related dyslipidemia will facilitate therapeutic choices that maintain viral suppression while reducing risk of atherosclerotic diseases. Apolipoprotein C-III (apoC-III) gene variants, which vary by race/ethnicity, have been associated with a lipid profile that resembles PI-induced dyslipidemia. However, the association of race/ethnicity, or candidate gene effects across race/ethnicity, with plasma lipid levels in HIV-1-infected individuals, has not been reported. Methods and Findings A cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and PI exposure on plasma lipids was performed in AIDS Clinical Trial Group studies (n = 626). Race/ethnicity was a highly significant predictor of plasma lipids in fully adjusted models. Furthermore, in stratified analyses, the effect of PI exposure appeared to differ across race/ethnicity. Black/non-Hispanic, compared with White/non-Hispanics and Hispanics, had lower plasma triglyceride (TG) levels overall, but the greatest increase in TG levels when exposed to PIs. In Hispanics, current PI antiretroviral therapy (ART) exposure was associated with a significantly smaller increase in TGs among patients with variant alleles at apoC-III-482, −455, and Intron 1, or at a composite apoC-III genotype, compared with patients with the wild-type genotypes. Conclusions In the first pharmacogenetic study of its kind in HIV-1 disease, we found race/ethnic-specific differences in plasma lipid levels on ART, as well as differences in the influence of the apoC-III gene on the development of PI-related hypertriglyceridemia. Given the multi-ethnic distribution of HIV-1 infection, our findings underscore the need for future studies of metabolic and cardiovascular complications of ART that specifically account for racial/ethnic heterogeneity, particularly when assessing candidate gene effects., This cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and protease inhibitor exposure on plasma lipids showed that race/ethnicity was a highly significant predictor of plasma lipids

We propose using mixed effects models to characterize the association between multiple gene polymorphisms, environmental factors and measures of disease progression. Characterizing high-order gene-gene and gene-environment interactions presents an analytic challenge due to the large number of candidate genes and the complex, undescribed interactions among them. Several approaches have been proposed recently to reduce the number of candidate genes and post hoc approaches to identify gene-gene interactions are described. However, these approaches may be inadequate for identifying high-order interactions in the absence of main effects and generally do not permit us to control for potential confounders. We describe how mixed effects models and related testing procedures overcome these limitations and apply this approach to data from a cohort of subjects at risk for cardiovascular disease. Four (4) genetic polymorphisms in three genes of the same gene family are considered. The proposed modelling approach allows us first to test whether there is a significant genetic contribution to the variability observed in our disease outcome. This contribution may be through main effects of multi-locus genotypes or through an interaction between genotype and environmental factors. This approach also enables us to identify specific multi-locus genotypes that interact with environmental factors in predicting the outcome. Mixed effects models provide a flexible statistical framework for controlling for potential confounders and identifying interactions among multiple genes and environmental factors that explain the variability in measures of disease progression.

Peripheral arterial disease is prevalent, associated with increased cardiovascular morbidity and mortality, and is underdiagnosed. Nevertheless, systematic efforts to provide early office-based peripheral arterial disease detection are not routinely implemented in office practice. The PARTNERS Program implemented the ankle brachial index (ABI) measurement in primary care outpatient clinics in order to model practical dissemination of this technique and thus improve office-based peripheral arterial disease detection. The objective of this study was to identify clinician-defined factors that were perceived to foster acceptance of, or create barriers to, the use of the ABI in office practice. The ABI Utilization Survey was administered to primary care clinicians who participated in the PARTNERS Program, as well as to additional primary care clinicians who participated in the PARTNERS Preceptorship. The survey assessed six parameters: pre- and post-participation office ABI utilization; perceived clinical utility of the ABI; perceived value of the ABI data relative to other commonly used office disease detection methods; feasibility of implementing office-based ABI testing; definition of factors limiting utilization of the ABI in office practice; and the role of office staff in performing the ABI test. Survey data were obtained from 886 respondents. A total of 68% of respondents did not measure the ABI prior to participation in the PARTNERS Program. After Program participation, the frequency of office ABI use increased from 12% to 43% weekly and 13% to 39% monthly. The few participants who reported using the ABI only once a year (annually) did not significantly change after the program. Most clinicians believed that the ABI was useful in the diagnosis and management of both symptomatic (96%) and asymptomatic (89%) peripheral arterial disease. Moderate to major barriers to use of the ABI included time constraints (56%), lack of reimbursement (45%), and staff availability (45%). Nearly all (88%) clinicians believed that it was feasible to incorporate ABI into daily practice. Overall, most clinicians (57 75%) believed that ABI was equal to, or more useful, than other widely available and reimbursed screening tests in preserving their patients’ health. In conclusion, the ABI was perceived by primary care clinicians to be a clinically useful diagnostic test. Limited reimbursement and time were identified as the primary barriers to its widespread use. Once learned, most clinicians stated that the ABI would continue to be frequently used in their office practice. The ABI is a simple peripheral arterial disease detection tool that can be successfully applied in primary care office practices.

Cigarette smoking activates platelets, induces endothelial inflammation, and promotes vascular dysfunction leading to accelerated atherosclerosis, plaque rupture, acute coronary syndromes, and death. Over several decades, notable advances in public policy in the United States have reduced initiation and increased cessation of cigarette smoking, thus lowering the prevalence of tobacco product consumption and related cardiovascular disease (CVD). Indeed, most people in developed countries understand the harm of cigarette smoking and the health and economic benefits of abstinence and cessation. However, these public health successes have slowed recently,1 reflecting perhaps a higher degree of addiction in remaining smokers and also a degree of fatigue in further advancing preventive and treatment strategies, as well as scientific knowledge more generally. Several realities underscore the current challenges …

Complex diseases such as cardiovascular disease are likely due to the effects of high-order interactions among multiple genes and demographic factors. Therefore, in order to understand their underlying biological mechanisms, we need to consider simultaneously the effects of genotypes across multiple loci. Statistical methods such as multifactor dimensionality reduction (MDR), the combinatorial partitioning method (CPM), recursive partitioning (RP), and patterning and recursive partitioning (PRP) are designed to uncover complex relationships without relying on a specific model for the interaction, and are therefore well-suited to this data setting. However, the theoretical overlap among these methods and their relative merits have not been well characterized. In this paper we demonstrate mathematically that MDR is a special case of RP in which (1) patterns are used as predictors (PRP), (2) tree growth is restricted to a single split, and (3) misclassification error is used as the measure of impurity. Both approaches are applied to a case-control study assessing the effect of eleven single nucleotide polymorphisms on coronary artery calcification in people at risk for cardiovascular disease.

Objective— Circulating levels of C-reactive protein (CRP) predict cardiovascular events. In contrast, an association between CRP and direct measures of atherosclerosis has not been established clearly. In the largest study to date, we examined the association of plasma CRP with coronary artery calcification (CAC) in 914 asymptomatic subjects in the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA). Methods and Results— In age-adjusted, cross-sectional analysis, there was a weak association between plasma CRP levels and CAC in women (odds ratio [OR] for ordinal regression, 1.1 [1.04 to 1.17] per 1.0 mg/L increase in CRP; P =0.005) but not in men. The association between CRP and CAC in women remained significant after adjusting for traditional risk factors (OR, 1.08 [1.00 to 1.14]; P =0.048) but was lost after further adjustment for body mass index (BMI) (OR, 1.02 [0.94 to 1.08]; P =0.7). Conclusions— In SIRCA, CRP was not associated with CAC in men, and a weak association in women was lost after adjustment for BMI. The relation between CRP and clinical events might not be related to atherosclerotic burden. Measures of inflammation, such as CRP, and indices of atherosclerosis, such as CAC, are likely to provide distinct information regarding cardiovascular risk.

OBJECTIVES This study was designed to determine if non-aspirin non-steroidal anti-inflammatory drugs (NANSAIDs) are associated with lower odds of myocardial infarction (MI) and if NANSAIDs, particularly ibuprofen, interfere with aspirin's cardioprotective effect. BACKGROUND The NANSAIDs may reduce the risk of MI but may also interfere with aspirin's cardioprotective effect. METHODS A case-control study was conducted, with cases of first, nonfatal MI identified prospectively and controls identified randomly from the community. RESULTS The use of NANSAIDs was associated with a significant reduction in MI among those not using aspirin (adjusted odds ratio [OR] 0.53; 95% confidence interval [CI]: 0.42 to 0.67). This was true for both ibuprofen (adjusted OR 0.52; 95% CI: 0.39 to 0.69) and naproxen (adjusted OR 0.48; 95% CI: 0.28 to 0.82). Although aspirin itself was associated with decreased odds of MI in those not also using NANSAIDs (adjusted OR relative to no aspirin use 0.79; 95% CI: 0.63 to 0.98), it was not associated with decreased odds of MI among those who were using NANSAIDs (OR 1.28; 95% CI: 0.85 to 1.94; p value for interaction = 0.026). The association of aspirin and reduced odds of MI diminished with increasing frequency of NANSAID use (test for interaction p = 0.006), particularly for ibuprofen (p = 0.018). Among frequent (4 times/week) NANSAID users, the OR for aspirin versus no aspirin was 2.04 (95% CI: 1.06 to 3.94). Users of prophylactic aspirin plus frequent ibuprofen had an OR relative to aspirin-only users of 2.03 (95% CI: 0.60 to 6.84). CONCLUSIONS In the absence of aspirin use, NANSAIDs are associated with reduced odds of MI. In those using aspirin, NANSAIDs do not provide additional protection. Additional study is needed to determine the clinical impact of using NANSAIDs along with aspirin for cardioprotection.

The risk of cardiovascular disease (CVD) is two- to fourfold greater in type 2 diabetics than in non-diabetics and cannot be accounted for by traditional risk factors alone. Coronary artery calcification (CAC) at electron beam computed tomography (EBCT) is a non-invasive index of coronary atherosclerosis. We hypothesized that the presence and extent of CAC would be greater in asymptomatic type 2 diabetics than in non-diabetics independent of traditional risk factors.We reviewed CAC data of all asymptomatic subjects referred for EBCT between 1996-1999 and compared CAC scores in type 2 diabetics ( n= 71) to all non-diabetics ( n= 1481) and to a randomly selected group of non-diabetics matched for all traditional CVD risk factors ( n= 71).CAC scores were greater in type 2 diabetics (272 +/- 472, median 41) than in all non-diabetics (104 +/- 288, median 4;0.01) and matched non-diabetics (188 +/- 354;0.05, median 12;0.05). The odds ratio (OR) for the presence of CAC (scores0) in type 2 diabetics was 2.9 [95% confidence intervals (CI) 1.1-7.8] after adjustment for traditional CVD risk factors. Type 2 diabetes was also associated (adjusted OR 2.15, 95%CI 1.3-3.6) with the extent of CAC when categorized as an ordinal outcome (CAC scores 0, 1-79, 80-399 and400). In type 2 diabetics, age, sex and body mass index were associated with extent of CAC.CAC scores at EBCT are greater in type 2 diabetics than non-diabetic subjects, cannot be accounted for by traditional risk factors alone and may be useful for identifying novel factors for coronary atherosclerosis in type 2 diabetes.

Correctly estimating isoform-specific gene expression is important for understanding complicated biological mechanisms and for mapping disease susceptibility genes. However, estimating isoform-specific gene expression is challenging because various biases present in RNA-Seq (RNA sequencing) data complicate the analysis, and if not appropriately corrected, can affect isoform expression estimation and downstream analysis. In this article, we present PennSeq, a statistical method that allows each isoform to have its own non-uniform read distribution. Instead of making parametric assumptions, we give adequate weight to the underlying data by the use of a non-parametric approach. Our rationale is that regardless what factors lead to non-uniformity, whether it is due to hexamer priming bias, local sequence bias, positional bias, RNA degradation, mapping bias or other unknown reasons, the probability that a fragment is sampled from a particular region will be reflected in the aligned data. This empirical approach thus maximally reflects the true underlying non-uniform read distribution. We evaluate the performance of PennSeq using both simulated data with known ground truth, and using two real Illumina RNA-Seq data sets including one with quantitative real time polymerase chain reaction measurements. Our results indicate superior performance of PennSeq over existing methods, particularly for isoforms demonstrating severe non-uniformity. PennSeq is freely available for download at http://sourceforge.net/projects/pennseq.

A retrospective, transesophageal study of 51 consecutive patients receiving a left ventricular (LV) assist device (AD) over a 2-year period showed that LVAD-associated LV thrombosis (16%) was predicted by acute myocardial infarction, atrial cannulation, and postimplantation bleeding, and was associated with a fourfold increased risk of stroke compared with patients without thrombosis. LV cannulation, when using short-term LVADs, may decrease the incidence of LV thrombosis, and early transition to Heartmate-LVAD support may improve outcome.

Adult domestic cats (Felis catus) appear to have the most polymorphic beta-globin system of any species. Reversed-phase high-performance liquid chromatography (RP-HPLC) revealed one alpha-chain and six different beta-globin chains. Each cat may have one to four different beta-globins, and a total of 17 different beta-globin patterns were identified. Based on family studies, a beta-globin gene region with two linked beta-globin gene loci and two to five alleles was proposed. In order to further define the molecular basis of this polymorphism we performed Southern blot analysis of the beta-globin gene region from 25 cats with 13 different HPLC patterns. Genomic DNA was digested with five restriction endonucleases (BamHI, BglII, EcoRI, HindIII, and PstI) and blots were hybridized with a human beta-globin probe. Restriction fragment length polymorphisms (RFLPs) with one to five fragments of 2.2-23 kb in size were found with BglII, EcoRI, HindIII, and PstI. Cats with specific HPLC beta-globin patterns had a unique DNA restriction pattern. The similarly sized BamHI and HindIII fragments of 4.6 kb suggest the presence of two closely linked genes. Furthermore, family studies suggest an autosomal codominant mode of inheritance of the beta-globin chains with seven haplotypes. Thus the RFLP data analyzed in the context of the HPLC haplotypes provide evidence at the DNA level for a feline beta-globin gene region with two closely linked gene loci and two to five alleles.

Oxidative stress is believed to play an important pathophysiological role in a variety of cardiovascular disorders, including atherosclerosis (1) and ischemia-reperfusion injury (2). Epidemiological studies have related the incidence of certain diseases inversely to the estimated intake of dietary antioxidants (3,4). Furthermore, data from certain animal models of oxidative stress lend indirect support for this hypothesis (5,6). However, difficulty in assessing oxygen radical generation in vivo has proven to be the major limitation to the understanding of this mechanism of human disease. Traditional in vitro assays of malondialdehyde and lipid hydroperoxides are thought to be fallible when applied to clinical investigation because of such factors as ex vivo generation of products, inaccuracy of assay methodology, and the instability and nonspecificity of the analytes (7,8). Alternative approaches rely on the measurement of indices of free radical generation explicitly induced ex vivo, such as copper-induced low-density lipoprotein (LDL) oxidation and the generation of spin traps (9,10). However, it is unclear how indices of LDL oxidizability relate to oxidant stress in vivo. Consequently, the relationship of oxidant stress to human disease remains unclear. Furthermore, there is little information on the actual in vivo antioxidant effects of vitamin C or E in humans. Thus, there is little biochemical basis for choice of disease and dose selection when such compounds are studied in phase III clinical trials (11,12).

Background —F 2 isoprostanes are stable, free radical–catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo. Methods and Results —Specific assays were developed by use of mass spectrometry for the F 2 isoprostanes iPF 2α -III and iPF 2α -VI and arachidonic acid (AA). Urinary excretion of the 2 F 2 isoprostanes was significantly increased in hypercholesterolemic patients, whereas substrate AA in urine did not differ between the groups. iPF 2α -III (pmol/mmol creatinine) was elevated ( P 2α -VI (281±22 versus 175±13; P 2α -III ( r =0.41; P 2α -VI ( r =0.39; P 2α -III (81±10 versus 59±4; P 2α -VI (195±18 versus 149±20; P 2α -III and iPF 2α -VI was correlated ( r =0.57; P 2α -III levels (ng/mg arachidonate) were elevated ( P 2 -III in LDL and urine were significantly correlated ( r =0.42; P Conclusions —Asymptomatic patients with moderate and severe hypercholesterolemia have evidence of oxidant stress in vivo.

The importance of thrombosis in acute coronary syndromes has prompted the widespread application of antithrombotic drugs and thrombolytic agents. Clearly, atherosclerosis provides a substrate for the development of arterial thrombosis. This has been viewed as a mechanical event with atherosclerosis culminating in a weakened plaque that ruptures at high shear. Alternatively, there may be a more subtle change in the interaction between the vessel wall and blood whereby the normal antithrombotic function of the vasculature is perturbed. This would explain the development of coronary thrombosis at seemingly low levels of coronary stenosis in young patients. While thrombosis has been traditionally viewed as a complication of atherosclerosis, there is evidence that coagulation proteins and platelets partake in the development of atherosclerosis. Finally, it is possible that both disordered vasculature and a prothrombotic state occur simultaneously, resulting in patients developing thrombosis early on a background of coronary atherosclerosis. For example, smoking results in vessel wall injury and also induces platelet activation. In this chapter, we review the role of thrombosis in the course of coronary atherosclerosis. The major involvement of thrombosis provides a rationale for the development of new antithrombotics and their widespread application in the management of this disease.

The development of methods to measure specific isoprostanes affords a unique opportunity to investigate both the role of oxidant stress as a mechanism of disease in vivo and to select rational doses of putative antioxidant drugs and vitamins for evaluation in human disease. The ability to measure these compounds directly in situ at the site of their formation, to immunolocalize them to target cells in atherosclerotic plaque and other tissues (61) and to assess their biosynthesis non-invasively in urine promises to elucidate the role of lipid peroxidation in cardiovascular disease.

Recent advances in RNA sequencing (RNA-Seq) have enabled the discovery of novel transcriptomic variations that are not possible with traditional microarray-based methods. Tissue and cell specific transcriptome changes during pathophysiological stress in disease cases versus controls and in response to therapies are of particular interest to investigators studying cardiometabolic diseases. Thus, knowledge on the relationships between sequencing depth and detection of transcriptomic variation is needed for designing RNA-Seq experiments and for interpreting results of analyses. Using deeply sequenced Illumina HiSeq 2000 101 bp paired-end RNA-Seq data derived from adipose of a healthy individual before and after systemic administration of endotoxin (LPS), we investigated the sequencing depths needed for studies of gene expression and alternative splicing (AS). In order to detect expressed genes and AS events, we found that ∼100 to 150 million (M) filtered reads were needed. However, the requirement on sequencing depth for the detection of LPS modulated differential expression (DE) and differential alternative splicing (DAS) was much higher. To detect 80% of events, ∼300 M filtered reads were needed for DE analysis whereas at least 400 M filtered reads were necessary for detecting DAS. Although the majority of expressed genes and AS events can be detected with modest sequencing depths (∼100 M filtered reads), the estimated gene expression levels and exon/intron inclusion levels were less accurate. We report the first study that evaluates the relationship between RNA-Seq depth and the ability to detect DE and DAS in human adipose. Our results suggest that a much higher sequencing depth is needed to reliably identify DAS events than for DE genes.

Background Chromosome 9p21 variants have been strongly associated with coronary heart disease in genome-wide association studies, but questions remain on the mechanism of risk, specifically whether the locus contributes to coronary atheroma burden or plaque instability. We investigated the relationship of 9p21 locus with (1) angiographic coronary artery disease (CAD) burden and (2) myocardial infarction (MI) in individuals with underlying CAD. Methods:We established a collaboration of 21 studies consisting of 33,673 subjects with information on both CAD (clinical or angiographic) and MI status along with 9p21 genotype. Tabular data were provided for each cohort on the presence and burden of angiographic CAD; MI cases with underlying CAD; and the diabetic status of all subjects. Results We first confirmed an association between 9p21 and CAD using angiographically defined cases and controls (pooled OR=1.31 (95% CI 1.20 to 1.43)). Among subjects with angiographic CAD (n=20,987), random-effects model identified an association with multi-vessel CAD, compared to those with single-vessel disease (OR=1.10 (95% CI 1.04 to 1.17) per copy of risk allele). Genotypic models showed an OR of 1.15 (95% CI 1.04 to 1.26) for heterozygous carrier and 1.23 (95% CI 1.08 to 1.39) for homozygous carrier. Finally, there was no significant association between 9p21 and prevalent MI when both cases (n=17 791) and controls (n=15 882) had underlying CAD (OR=0.99 (95% CI 0.95 to 1.03) per risk allele). Conclusions The 9p21 locus shows convincing association with greater burden of CAD, but not with MI in the presence of underlying CAD. This adds further weight to the hypothesis that 9p21 locus primarily mediates an atherosclerotic phenotype.

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10−8–1.2×10−43). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p, Author Summary Serum adiponectin levels are highly heritable and are inversely correlated with the risk of type 2 diabetes (T2D), coronary artery disease, stroke, and several metabolic traits. To identify common genetic variants associated with adiponectin levels and risk of T2D and metabolic traits, we conducted a meta-analysis of genome-wide association studies of 45,891 multi-ethnic individuals. In addition to confirming that variants at the ADIPOQ and CDH13 loci influence adiponectin levels, our analyses revealed that 10 new loci also affecting circulating adiponectin levels. We demonstrated that expression levels of several genes in these candidate regions are associated with serum adiponectin levels. Using a powerful novel method to assess the contribution of the identified variants with other traits using summary-level results from large-scale GWAS consortia, we provide evidence that the risk alleles for adiponectin are associated with deleterious changes in T2D risk and metabolic syndrome traits (triglycerides, HDL, post-prandial glucose, insulin, and waist-to-hip ratio), demonstrating that the identified loci, taken together, impact upon metabolic disease.

Papers p 71 Education and debate p 103 The efficacy of aspirin in the secondary prevention of myocardial infarction and stroke is widely accepted. The evidence which supports this perception includes its identification as an inhibitor of cyclo-oxygenase and platelet aggregation; identification of the major product of platelet cyclo-oxygenase as thromboxane A2, a vasoconstrictor and platelet agonist; the discovery that aspirin irreversibly acetylates cyclo-oxygenase, permitting cumulative inhibition by low doses of thromboxane A2 formation in the presystemic circulation; the discovery that thromboxane A2 biosynthesis is increased during ischaemic episodes of unstable angina; and the demonstration in individual, controlled, prospective double blind trials that aspirin reduces both myocardial infarction and death in unstable angina by 50%, whether given at 75 mg, 324 mg, or 1300 mg/day. 1 2 Following these discoveries Collins, Peto, Baigent, and their colleagues in Oxford organised the Antithrombotic Trialists' Collaboration to share data and permit overview analyses of controlled trials of antiplatelet drugs. At the time of the initial reports, these trials mainly involved aspirin and confirmed its efficacy in syndromes of acute vascular occlusion such as unstable angina, while suggesting a net benefit in the secondary prevention of stroke.3–5 Today, Baigent and colleagues report further analyses (p 71),6 though a critic …

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., Author Summary To date, most large-scale genome-wide association studies (GWAS) carried out to identify risk factors for complex human diseases and traits have focused on population of European ancestry. It is currently unknown whether the same loci associated with complex diseases and traits in Caucasians will replicate in population of African ancestry. Here, we conducted a large GWAS to identify common DNA polymorphisms associated with coronary heart disease (CHD) and its risk factors (type-2 diabetes, hypertension, smoking status, and LDL- and HDL-cholesterol) in 8,090 African Americans as part of the NHLBI Candidate gene Association Resource (CARe) Project. We replicated 17 associations previously reported in Caucasians, suggesting that the same loci carry common DNA sequence variants associated with CHD and its risk factors in Caucasians and African Americans. At five of these 17 loci, we used the different patterns of linkage disequilibrium between populations of European and African ancestry to identify DNA sequence variants more strongly associated with phenotypes than the index SNPs found in Caucasians, suggesting smaller genomic intervals to search for causal alleles. We also used the CARe data to develop new statistical methods to perform association studies in admixed populations. The CARe Project data represent an extraordinary resource to expand our understanding of the genetics of complex diseases and traits in non-European-derived populations.

We acknowledge the comments made by Stefan et al. (1) and appreciate the opportunity to address them further while clarifying some points. The Penn Diabetes Heart Study (PDHS) is not a retrospective study but a prospective community-based cohort focusing on subclinical atherosclerosis in patients with type 2 diabetes. Notably, PDHS participants are recruited specifically to exclude clinical cardiovascular disease (CVD) or moderate-to-severe chronic kidney disease (CKD). However, the substantial burden of subclinical coronary atherosclerosis observed in PDHS participants (2) suggests an increased long-term cardiovascular risk. Therefore, the PDHS provides the opportunity to study subclinical peripheral atherosclerosis in patients with type 2 diabetes without the confounding and possible reverse causation associated with …

Objective— Genetic studies might provide new insights into the biological mechanisms underlying lipid metabolism and risk of CAD. We therefore conducted a genome-wide association study to identify novel genetic determinants of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides. Methods and Results— We combined genome-wide association data from 8 studies, comprising up to 17 723 participants with information on circulating lipid concentrations. We did independent replication studies in up to 37 774 participants from 8 populations and also in a population of Indian Asian descent. We also assessed the association between single-nucleotide polymorphisms (SNPs) at lipid loci and risk of CAD in up to 9 633 cases and 38 684 controls. We identified 4 novel genetic loci that showed reproducible associations with lipids (probability values, 1.6×10 −8 to 3.1×10 −10 ). These include a potentially functional SNP in the SLC39A8 gene for HDL-C, an SNP near the MYLIP/GMPR and PPP1R3B genes for LDL-C, and at the AFF1 gene for triglycerides. SNPs showing strong statistical association with 1 or more lipid traits at the CELSR2 , APOB , APOE-C1-C4-C2 cluster, LPL , ZNF259-APOA5-A4-C3-A1 cluster and TRIB1 loci were also associated with CAD risk (probability values, 1.1×10 −3 to 1.2×10 −9 ). Conclusion— We have identified 4 novel loci associated with circulating lipids. We also show that in addition to those that are largely associated with LDL-C, genetic loci mainly associated with circulating triglycerides and HDL-C are also associated with risk of CAD. These findings potentially provide new insights into the biological mechanisms underlying lipid metabolism and CAD risk.

Purpose A convergence of innate immune and adipose signals is thought to promote metabolic syndrome and atherosclerosis. Indeed, septicemia and experimental endotoxemia induce insulin resistance and metabolic dyslipidemia. However, the role of innate immunity in regulating adipokines, key modulators of insulin resistance and atherosclerosis, is poorly defined in humans. Method A human endotoxemia model was used to examine the impact of activation of innate immunity on resistin, adiponectin and leptin pathways in vivo. Serial whole blood and adipose (subcutaneous biopsies) gene expression (quantitative PCR) as well as plasma levels of adipokines were measured for 24 hours prior to and following intravenous administration of 3 ng/kg endotoxin (LPS) in 20 healthy human volunteers (50% male, 80% Caucasian, mean age 27.3 ± 4.8, inpatient GCRC protocol). Repeated measures analysis of variance was applied to the data. Findings Whole blood resistin mRNA levels (13.7 ± 7.3 fold; F statistic = 47.3, p Conclusions Endotoxemia induces specific and differential effects on adipokine signaling in humans. Strikingly, resistin appears to be a leukocyte, but not adipose, derived inflammatory cytokine in humans, which is vastly increased in response to LPS. In contrast, adiponectin levels are unchanged during endotoxemia, but expression of both adiponectin receptor subtypes is decreased, consistent with an attenuation of adiponectin insulin sensitizing and anti-atherosclerotic signaling in vivo.

Purpose A convergence of inflammatory and adipose signals has been proposed as a mechanism for metabolic syndrome and atherosclerosis. We hypothesized that adipose tissue may influence the response to acute inflammation in vivo in humans. Method As part of a sixty-hour GCRC protocol of 20 healthy volunteers [50% male, 80% Caucasian, mean age 27.3 ± 4.8; 10 lean (BMI = 20.55 ± 1.75) and 10 overweight (BMI = 27.08 ± 0.88)], we examined peak responses in serial whole blood and adipose (subcutaneous fat biopsies) gene expression, and plasma hs-C reactive protein (hs-CRP) following intravenous administration of 3 ng/kg human research grade endotoxin. Findings As expected, we observed increases (median fold increase and inter-quartile range) in whole blood mRNA expression of TNF alpha [8.8 fold (6.2-12.0)], IL-6 [3.8 (1.7-7.5)] and IL-10 [5.1 (2.4-7.7)], as well as plasma hs-CRP levels (mean peak hs-CRP 44.5 ± 21.7) following intravenous endotoxin (p Conclusions Our findings demonstrate, for the first time, that human adipose gene expression is regulated by activation of innate immunity in vivo, and preliminary analyses suggest that the response to an inflammatory stimulus may be influenced by the degree of adiposity. These observations provide novel insight into the role of adipose in promoting pro-atherosclerotic inflammatory responses in humans.

Studies have postulated that cyclooxygenase-2 (COX-2) selective inhibitors affect cardiovascular risk through various mechanisms. Some of these mechanisms could increase risk (for example, inhibition of prostacyclin production), and some could decrease risk (for example, inhibition of inflammation).To determine the effect of COX-2 inhibitors on risk for nonfatal myocardial infarction (MI).Case-control study.36 hospitals in a 5-county area.1718 case-patients with a first, nonfatal MI admitted to these hospitals and 6800 controls randomly selected from the same counties.Self-reported medication use assessed through telephone interviews.The adjusted odds ratio for MI among celecoxib users, relative to persons who did not use nonaspirin nonsteroidal anti-inflammatory drugs (NSAIDs), was 0.43 (95% CI, 0.23 to 0.79) compared with 1.16 (CI, 0.70 to 1.93) among rofecoxib users. The use of rofecoxib was associated with a statistically significant higher odds of MI compared with the use of celecoxib (adjusted odds ratio for rofecoxib vs. celecoxib, 2.72 [CI, 1.24 to 5.95]; P = 0.01). Nonselective NSAIDs were associated with a reduced odds of nonfatal MI relative to nonusers. Comparisons of COX-2 inhibitors with nonselective NSAIDs were the following: rofecoxib versus naproxen (odds ratio, 3.39 [CI, 1.37 to 8.40]) and celecoxib versus ibuprofen or diclofenac (odds ratio, 0.77 [CI, 0.40 to 1.48]).The possibility of recall bias and uncontrolled confounding in this observational study limit the ability to make definitive conclusions. The association of celecoxib with a lower odds of MI could have occurred by chance. Only about 50% of eligible participants completed telephone interviews.Celecoxib and rofecoxib were associated with different odds of MI. Cardiovascular effects among the COX-2 inhibitors seem different, but further studies, preferably randomized trials, are needed to fully understand the spectrum of effects of COX-2 inhibitors and potential differences among them.

Three families of English springer spaniel dogs with phosphofructokinase (PFK) deficiency causing haemolysis were studied. Four male dogs and one female dog with chronic haemolysis and haemolytic crises were found to have markedly reduced PFK activity in erythrocytes (8-20% of control English springer spaniels). PFK-deficient erythrocytes exhibited an extreme alkaline and sucrose lysis. The oxygen dissociation curve of erythrocyte suspensions was shifted to the left with a 50% saturation of haemoglobin at a partial oxygen pressure of 16-17 mmHg (normal 26-31 mmHg). Muscle wasting and mildly increased serum creatine phosphokinase activity were also noted. Six clinically normal first degree relatives of affected dogs had erythrocyte PFK activities that were 38-51% of controls. In these family members, there was an erythrocytosis and mild reticulocytosis probably due to a mildly enhanced haemoglobin-oxygen affinity but no increase in serum creatine phosphokinase. These studies confirm the familial nature of muscle-type PFK deficiency in English springer spaniels and support the conclusion that this animal model of the human glycogen storage disease type VII is inherited as an autosomal recessive trait.

Objectives We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455), and clinical coronary artery disease (CAD). Background Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with noncarriers. Methods The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in 19 case-control studies of nonfatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports. Results A total of 17,000 cases and 39,369 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the 19 studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with noncarriers. Regression analyses and fixed-effects meta-analyses ruled out with high degree of confidence an increase of >= 2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early-onset disease (younger than 50 years of age for men and younger than 60 years of age for women) compared with similarly aged controls as well as all non-European subgroups. Conclusions The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study. (J Am Coll Cardiol 2010;56:1552-63) (C) 2010 by the American College of Cardiology Foundation

Interindividual variation in mean leukocyte telomere length (LTL) is associated with cancer and several age-associated diseases. We report here a genome-wide meta-analysis of 37,684 individuals with replication of selected variants in an additional 10,739 individuals. We identified seven loci, including five new loci, associated with mean LTL (P

Background Coronary heart disease (CHD) is the major cause of death in the United States. Coronary artery calcification (CAC) scores are independent predictors of CHD. African Americans (AA) have higher rates of CHD but are less well-studied in genomic studies. We assembled the largest AA data resource currently available with measured CAC to identify associated genetic variants. Methods We analyzed log transformed CAC quantity (ln(CAC + 1)), for association with ~2.5 million single nucleotide polymorphisms (SNPs) and performed an inverse-variance weighted meta-analysis on results for 5,823 AA from 8 studies. Heritability was calculated using family studies. The most significant SNPs among AAs were evaluated in European Ancestry (EA) CAC data; conversely, the significance of published SNPs for CAC/CHD in EA was queried within our AA meta-analysis. Results Heritability of CAC was lower in AA (~30%) than previously reported for EA (~50%). No SNP reached genome wide significance (p

Height is a classic complex trait with common variants in a growing list of genes known to contribute to the phenotype. Using a genecentric genotyping array targeted toward cardiovascular-related loci, comprising 49,320 SNPs across approximately 2000 loci, we evaluated the association of common and uncommon SNPs with adult height in 114,223 individuals from 47 studies and six ethnicities. A total of 64 loci contained a SNP associated with height at array-wide significance (p < 2.4 × 10-6), with 42 loci surpassing the conventional genome-wide significance threshold (p < 5 × 10-8). Common variants with minor allele frequencies greater than 5% were observed to be associated with height in 37 previously reported loci. In individuals of European ancestry, uncommon SNPs in IL11 and SMAD3, which would not be genotyped with the use of standard genome-wide genotyping arrays, were strongly associated with height (p < 3 × 10-11). Conditional analysis within associated regions revealed five additional variants associated with height independent of lead SNPs within the locus, suggesting allelic heterogeneity. Although underpowered to replicate findings from individuals of European ancestry, the direction of effect of associated variants was largely consistent in African American, South Asian, and Hispanic populations. Overall, we show that dense coverage of genes for uncommon SNPs, coupled with large-scale meta-analysis, can successfully identify additional variants associated with a common complex trait. © 2011 The American Society of Human Genetics.

To the Editor: The role of niacin in the era of widespread statin use has been called into question by 2 recent clinical trials, AIM-HIGH (Atherothrombosis Intervention in Metabolic Syndrome With Low HDL/High Triglycerides: Impact on Global Health) and HPS2-THRIVE (Treatment of HDL to Reduce the