Tag polymorphisms at the A20 (TNFAIP3) locus are associated with lower gene expression and increased risk of coronary artery disease in type 2 diabetes.

A20 or tumor necrosis factor (TNF)-induced protein 3 (TNFAIP3) is a negative regulator of nuclear factor-kappaB (NF-kappaB). We have investigated whether polymorphisms in this gene are associated with increased atherosclerosis in diabetic patients. Five tag single nucleotide polymorphisms (SNPs) were typed in 479 type 2 diabetic patients from Boston, including 239 coronary artery disease (CAD)-positive case subjects and 240 CAD-negative control subjects. Two tag SNPs (rs5029930 and rs610604) were independently associated with CAD; adjusted odds ratios (ORs) for minor allele carriers were 2.3 (95% CI 1.4-3.8, P = 0.001) and 2.0 (1.3-2.9, P = 0.0008), respectively. The association with rs610604 was dependent on glycemic control, with ORs of 3.9 among subjects with A1C < or =7.0% and 1.2 for those with A1C >7.0% (P for interaction = 0.015). A similar interaction pattern was found among 231 CAD-positive and 332 CAD-negative type 2 diabetic patients from Italy (OR 2.2, P = 0.05 vs. OR 0.9, P = 0.63 in the low vs. high A1C strata, P for interaction = 0.05). Quantitative RT-PCR in blood mononuclear cells from 83 nondiabetic subjects showed that rs610604 and rs5029930 minor allele homozygotes have 30-45% lower levels of A20 mRNA than major allele homozygotes, and heterozygotes have intermediate levels (P = 0.04 and 0.028, respectively). These findings point to variability in the A20/TNFAIP3 gene as a modulator of CAD risk in type 2 diabetes. This effect is mediated by allelic differences in A20 expression.

Interaction between poor glycemic control and 9p21 locus on risk of coronary artery disease in type 2 diabetes.

CONTEXT: A common allele on chromosome 9p21 has been repeatedly associated with increased risk of coronary artery disease (CAD) in the general population. However, the magnitude of this effect in the population with diabetes has not been well characterized. OBJECTIVE: To examine the association of the 9p21 variant with CAD in individuals with type 2 diabetes and evaluate its interaction with poor glycemic control. DESIGN, SETTING, AND PARTICIPANTS: (1) Case-control study of 734 type 2 diabetes patients (322 with angiographically diagnosed CAD and 412 with no evidence of CAD) who were recruited between 2001 and 2006 at the Joslin Clinic, Beth Israel Deaconess Medical Center; and (2) independent cohort study of 475 type 2 diabetes patients from the Joslin Clinic whose survival status was monitored from their recruitment between 1993 and 1996 until December 31, 2004. Participants for both studies were genotyped for a representative single-nucleotide polymorphism at 9p21 (rs2383206) and characterized for their long-term glycemic control by averaging multiple hemoglobin A(1c) (HbA(1c)) measurements taken in the years before study entry. MAIN OUTCOME MEASURES: For the case-control study, association between single-nucleotide polymorphism rs2383206 and CAD defined as angiographically documented stenosis greater than 50% in a major coronary artery or a main branch thereof was assessed and for the cohort study, cumulative 10-year mortality was documented. RESULTS: Individuals who were homozygous for the risk allele were significantly more frequent among case than control participants (42.3% vs 28.9P = .0002). This association was unaffected by adjustment for cardiovascular risk factors, but the effect of the risk genotype was significantly magnified (adjusted P for interaction = .048) in the presence of poor glycemic control (worst tertile of the distribution of HbA(1c) at examination). Relative to the CAD risk for patients with neither a 9p21 risk allele nor poor glycemic control, the CAD odds for participants having 2 risk alleles but not poor glycemic control were increased 2-fold (odds ratio [OR], 1.99; 95% confidence interval [CI], 1.17-3.41), whereas the odds for study participants with the same genotype and with poor glycemic control were increased 4-fold (OR, 4.27; 95% CI, 2.26-8.01). The interaction was stronger (adjusted P = .005) when a measure of long-term glycemic control (7-year average rather than most recent HbA(1c)) was used with ORs of 7.83 (95% CI, 3.49-17.6) for participants having 2 risk alleles and a history of poor glycemia and 1.54 (95% CI, 0.72-3.30) for participants with the same genotype but without this exposure. A similar interaction between 9p21 variant and poor glycemic control was observed with respect to cumulative 10-year mortality in the cohort study (43.6% in patients with 2 risk alleles and poor glycemic control, 23.1% in individuals with only the 2 risk alleles, 30.0% in individuals with only poor glycemic control, and 31.6% in individuals with neither factor, P for interaction, = .036). CONCLUSION: In this study population, the CAD risk associated with the 9p21 variant was increased in the presence of poor glycemic control in type 2 diabetes.

Common haplotypes at the adiponectin receptor 1 (ADIPOR1) locus are associated with increased risk of coronary artery disease in type 2 diabetes.

Adiponectin, an adipokine facilitating insulin action, has antiatherogenic effects. This study investigated whether common polymorphisms in the adiponectin receptor 1 (ADIPOR1) gene mediating these effects influence the risk of coronary artery disease (CAD) in type 2 diabetes. Linkage disequilibrium analysis of 28 single nucleotide polymorphisms (SNPs) spanning the entire ADIPOR1 locus revealed two haplotype blocks that could be tagged by six SNPs. These six markers were typed in two populations of CAD-positive and -negative subjects with type 2 diabetes, one from Boston (n = 411) and the other from Italy (n = 533). In the Boston population, the three tags of the more 3' block were all significantly associated with CAD (P = 0.001-0.01). A similar trend, although not significant, was found in Italian subjects. Haplotype analysis of the combined populations revealed different haplotype distributions in case and control subjects (P = 0.0002), with one common haplotype being associated in homozygotes with a greater than threefold increase in cardiovascular risk (odds ratio 3.6 [95% CI 1.8-7.2]). Some of the genotypes associated with increased cardiovascular risk were associated with 30-40% lower ADIPOR1 mRNA levels in blood mononuclear cells (n = 60) and adipose tissue biopsies (n = 28) (P = 0.001-0.014). Our findings point to genetic variability at the ADIPOR1 locus as a strong determinant of CAD susceptibility in type 2 diabetes.

Genetic variability at the leptin receptor (LEPR) locus is a determinant of plasma fibrinogen and C-reactive protein levels.

OBJECTIVE: Cellular and animal studies suggest that leptin has proinflammatory and prothrombotic effects that could link increased adipose mass directly to atherogenesis. To investigate this hypothesis, we examined the effect of genetic variability at the leptin receptor (LEPR) locus on the plasma levels of fibrinogen and CRP--two markers of inflammation and susceptibility to atherosclerosis. METHODS AND RESULTS: Linkage disequilibrium analysis of 71 single-nucleotide polymorphisms (SNPs) spanning the LEPR locus revealed four haplotype blocks that could be tagged by 11 SNPs. In 630 healthy Caucasian individuals, variability in block #4 was significantly associated with plasma fibrinogen (p=0.005), accounting for 3% of its variance (r2=0.030). The same block was also associated with CRP levels (p=0.049, r2=0.022). The effect was strongest for two of the SNPs in this block. At rs3790432, fibrinogen was 10% higher in minor allele homozygotes than in major allele homozygotes and intermediate in heterozygotes (p=0.015). At rs1805096, it was 5% higher (p=0.007) and CRP 32% higher (p=0.011) in major allele homozygotes than in minor allele carriers. This pattern of association was also evident in the haplotype analysis. CONCLUSIONS: Association of leptin receptor variability with inflammatory traits supports the hypothesis that leptin may play a role in atherogenesis.

The effect of vitamin E on endothelial function of micro- and macrocirculation and left ventricular function in type 1 and type 2 diabetic patients.

We examined the effects of high-dosage vitamin E treatment over a 12-month period on the vascular reactivity of micro- and macrocirculation and left ventricular function in diabetic patients. Subjects (n = 89) were randomized to vitamin E (1,800 IU daily) or placebo and were followed for 12 months. High-resolution ultrasound images were used to measure the flow-mediated dilation (FMD; endothelium dependent) and nitroglycerin-induced dilation (NID; endothelium independent) of the brachial artery. Laser Doppler perfusion imaging was used to measure vascular reactivity in the forearm skin. Left ventricular function was evaluated using transthoracic echocardiogram. At the end of the 6-month period, a worsening in endothelium-dependent skin vasodilation (P = 0.02) and rise in endothelin levels (P = 0.01) were found in the vitamin E compared with the placebo group. At the end of the 12-month period, a worsening was observed in NID (P = 0.02) and a marginal worsening was seen in systolic blood pressure (P = 0.04) and FMD (P = 0.04) in the vitamin E compared with the placebo group. In addition C-reactive protein levels decreased marginally in the vitamin E compared with the placebo group (P = 0.05). No changes were observed in left ventricular function. We concluded that long-term treatment with 1,800 IU of vitamin E has no beneficial effects on endothelial or left ventricular function in diabetic patients. Because vitamin E-treated patients had a worsening in some vascular reactivity measurements when compared with control subjects, the use of high dosages of vitamin E cannot be recommended.

Angiotensin receptor blockade with candesartan attenuates atherosclerosis, plaque disruption, and macrophage accumulation within the plaque in a rabbit model.

BACKGROUND: Little is known about whether direct angiotensin receptor blockade can reduce atherosclerosis and plaque disruption. This study evaluated the effect of angiotensin receptor blockade on both the development of atherosclerosis and the disruption of plaque in a modified Constantinides animal model. METHODS AND RESULTS: Twenty-eight New Zealand White rabbits underwent aortic balloon injury followed by a 1% cholesterol diet for 8 weeks. Thirteen rabbits received candesartan at 0.5 mg x kg(-1) x d(-1) beginning 2 days before aortic balloon injury and continued for the total 8 weeks of the cholesterol diet. The rabbits were then pharmacologically triggered and humanely killed, and their aortas were analyzed. The degree of atherosclerosis was determined by intima-media ratio of the infrarenal portion of the aorta. The frequency of intra-aortic thrombosis, a measure of plaque disruption, and the percentages of macrophage area and collagen-staining area of the plaque were determined. Candesartan-treated rabbits had less atherosclerosis (intima-media infrarenal aorta ratio of 1.18+/-0.08 versus 1.57+/-0.08 [mean+/-SEM] for the placebo group, P<0.001); fewer thrombi (3 of 13 versus 11 of 15; P<0.05); lower percentage area of macrophages to total plaque (18.8+/-2.7% versus 27+/-2.5%, P<0.05); and higher collagen to total plaque area (45+/-3% versus 35+/-2%, P<0.01). CONCLUSIONS: These results demonstrate that angiotensin receptor blockade attenuates the degree of atherosclerosis and reduces both plaque disruption and macrophage accumulation while increasing collagen deposition in the aortas of this animal model.

In vivo molecular imaging of acute and subacute thrombosis using a fibrin-binding magnetic resonance imaging contrast agent.

BACKGROUND: Plaque rupture with subsequent thrombosis is recognized as the underlying pathophysiology of most acute coronary syndromes and stroke. Thus, direct thrombus visualization may be beneficial for both diagnosis and guidance of therapy. We sought to test the feasibility of direct imaging of acute and subacute thrombosis using MRI together with a novel fibrin-binding gadolinium-labeled peptide, EP-1873, in an experimental animal model of plaque rupture and thrombosis. METHODS AND RESULTS: Fifteen male New Zealand White rabbits (weight, approximately 3.5 kg) were made atherosclerotic by feeding a high-cholesterol diet after endothelial aortic injury. Plaque rupture was then induced with the use of Russell's viper venom (RVV) and histamine. Subsequently, MRI of the subrenal aorta was performed before RVV, after RVV, and after EP-1873. Histology was performed on regions suggested by MRI to contain thrombus. Nine rabbits (60%) developed plaque rupture and thrombus, including 25 thrombi visually apparent on MRI as "hot spots" after injection of EP-1873. Histological correlation confirmed all 25 thrombi (100%), with no thrombi seen in the other regions of the aorta. In the remaining 6 rabbits (control) without plaque rupture, no thrombus was observed on the MR images or on histology. CONCLUSIONS: We demonstrate the feasibility of in vivo "molecular" MRI for the detection of acute and subacute thrombosis using a novel fibrin-binding MRI contrast agent in an animal model of atherosclerosis and acute/subacute thrombosis. Potential clinical applications include thrombus detection in acute coronary syndromes and stroke.

A common haplotype at the CD36 locus is associated with high free fatty acid levels and increased cardiovascular risk in Caucasians.

CD36 is a class B scavenger receptor recognizing a variety of ligands including long-chain fatty acids and modified LDL. We investigated whether genetic variability at this locus is a determinant of free fatty acid (FFA) plasma levels and risk of coronary artery disease (CAD) in Caucasians. Typing of 21 polymorphic markers, evenly spanning the CD36 gene, revealed two linkage disequilibrium (LD) blocks that could be tagged by five polymorphisms (-33137A>G, -31118G>A, 25444G>A, 27645del>ins and 30294G>C). In 585 non-diabetic individuals of Caucasian origin, the 30294G>C polymorphism was significantly associated with FFA levels (P = 0.02)--an effect that was especially visible among men (P = 0.009). A similar association was observed in this gender at -33137 (P = 0.008) and -31118 (P = 0.028). When the five tag polymorphisms were considered together, men carrying the AGGIG haplotype had 31% higher FFA (P = 0.0002) and 20% higher triglycerides (P = 0.025) than non-carriers. The same haplotype was associated with increased risk of CAD in 197 type 2 diabetic individuals from the US (OR = 2.3, 95% CI 1.2-4.2). A similar tendency was observed in a group of 321 type 2 diabetic individuals from Italy (OR = 1.4, 0.9-2.3), resulting in an overall relative risk of 1.6 (1.1-2.3, P = 0.015) in the two populations considered together. By targeted resequencing, we identified a common variant in the CD36 promoter that is in strong LD with the AGGIG haplotype and could be partly responsible for these findings. In conclusion, this comprehensive study of CD36 variability indicates that the common polymorphisms at this locus modulate lipid metabolism and cardiovascular risk in Caucasians.