Ross C. Laxton

Advanced Glycation End-Product of Low Density Lipoprotein Activates the Toll-Like 4 Receptor Pathway Implications for Diabetic Atherosclerosis

Objective—Diabetes is a major risk factor for coronary heart disease. Accumulation of advanced glycation end-products (AGEs) attributable to hyperglycemia in diabetics promotes the development of atherosclerosis. However, the underlying mechanisms remain unclear. Methods and Results—The advanced glycation end-product of low-density-lipoprotein (AGE-LDL) induced proinflammatory cytokine production in human coronary artery endothelial cells and human- and mouse-macrophages. AGE-LDL stimulated cytokine synthesis was markedly reduced in mouse macrophages with a TLR4 loss-of-function mutation. Coimmunoprecipitation experiments indicated AGE-LDL interacts with TLR4, RAGE, and CD36. Incubation of cultured macrophages with TLR4, RAGE, or CD36 antibodies inhibited AGE-LDL stimulation of tumor necrosis factor (TNF)&agr; production. A competitive binding inhibitor of TLR4 blocked AGE-LDL binding to the receptor. After transfection of a HEK293 cell system with wild-type TLR4, AGE-LDL activated a signaling pathway including p38&agr;, JNK, and ERK1 kinases and AP1, Elk1, and NF–&kgr;B transcription factors; the net result being increased cytokine production. These effects were absent when cells were transfected with empty plasmid. Two common polymorphisms in TLR4, D299G and T399I, reduced the response of TLR4 to lipopolysaccharide (LPS) but had no effect on AGE-LDL signaling. Conclusions—These results indicate that AGE-LDL activates a TLR4-mediated signaling pathway, thus inducing proinflammatory cytokine production. This mechanism may partly explain the increased risk of atherosclerosis observed in diabetics.

A Role of Matrix Metalloproteinase-8 in Atherosclerosis

Rationale: Atherosclerotic lesions express matrix metalloproteinase (MMP)8, which possesses proteolytic activity on matrix proteins particularly fibrillar collagens and on nonmatrix proteins such as angiotensin (Ang) I. Objective: We studied whether MMP8 plays a role in atherogenesis. Methods and Results: In atherosclerosis-prone apolipoprotein E–deficient mice, inactivating MMP8 resulted in a substantial reduction in atherosclerotic lesion formation. Immunohistochemical examinations showed that atherosclerotic lesions in MMP8-deficient mice had significantly fewer macrophages but increased collagen content. In line with results of in vitro assays showing that Ang I cleavage by MMP8 generated Ang II, MMP8 knockout mice had lower Ang II levels and lower blood pressure. In addition, we found that products of Ang I cleavage by MMP8 increased vascular cell adhesion molecule (VCAM)-1 expression and that MMP8-deficient mice had reduced VCAM-1 expression in atherosclerotic lesions. Intravital microscopy analysis showed that leukocyte rolling and adhesion on vascular endothelium was reduced in MMP8 knockout mice. Furthermore, we detected an association between MMP8 gene variation and extent of coronary atherosclerosis in patients with coronary artery disease. A relationship among MMP8 gene variation, plasma VCAM-1 level, and atherosclerosis progression was also observed in a population-based, prospective study. Conclusions: These results indicate that MMP8 is an important player in atherosclerosis.

Comparative analysis of genome-wide association studies signals for lipids, diabetes, and coronary heart disease: Cardiovascular Biomarker Genetics Collaboration

Aims To evaluate the associations of emergent genome-wide-association study-derived coronary heart disease (CHD)-associated single nucleotide polymorphisms (SNPs) with established and emerging risk factors, and the association of genome-wide-association study-derived lipid-associated SNPs with other risk factors and CHD events. Methods and results Using two case–control studies, three cross-sectional, and seven prospective studies with up to 25 000 individuals and 5794 CHD events we evaluated associations of 34 genome-wide-association study-identified SNPs with CHD risk and 16 CHD-associated risk factors or biomarkers. The Ch9p21 SNPs rs1333049 (OR 1.17; 95% confidence limits 1.11–1.24) and rs10757274 (OR 1.17; 1.09–1.26), MIA3 rs17465637 (OR 1.10; 1.04–1.15), Ch2q36 rs2943634 (OR 1.08; 1.03–1.14), APC rs383830 (OR 1.10; 1.02, 1.18), MTHFD1L rs6922269 (OR 1.10; 1.03, 1.16), CXCL12 rs501120 (OR 1.12; 1.04, 1.20), and SMAD3 rs17228212 (OR 1.11; 1.05, 1.17) were all associated with CHD risk, but not with the CHD biomarkers and risk factors measured. Among the 20 blood lipid-related SNPs, LPL rs17411031 was associated with a lower risk of CHD (OR 0.91; 0.84–0.97), an increase in Apolipoprotein AI and HDL-cholesterol, and reduced triglycerides. SORT1 rs599839 was associated with CHD risk (OR 1.20; 1.15–1.26) as well as total- and LDL-cholesterol, and apolipoprotein B. ANGPTL3 rs12042319 was associated with CHD risk (OR 1.11; 1.03, 1.19), total- and LDL-cholesterol, triglycerides, and interleukin-6. Conclusion Several SNPs predicting CHD events appear to involve pathways not currently indexed by the established or emerging risk factors; others involved changes in blood lipids including triglycerides or HDL-cholesterol as well as LDL-cholesterol. The overlapping association of SNPs with multiple risk factors and biomarkers supports the existence of shared points of regulation for these phenotypes.

Association of Matrix Metalloproteinase-8 Gene Variation with Breast Cancer Prognosis

Animal and cell studies indicate an inhibitory effect of matrix metalloproteinase-8 (MMP8) on tumorigenesis and metastasis. We investigated whether MMP8 gene variation was associated with breast cancer metastasis and prognosis in humans. We first studied nine tagging single nucleotide polymorphisms (SNP) in the MMP8 gene in 140 clinically and pathologically well-characterized breast cancer patients. Four of the SNPs were found to be associated with lymph node metastasis, the most pronounced being a promoter SNP (rs11225395) with its minor allele (T) associating with reduced susceptibility to lymph node metastasis (P = 0.02). This SNP was further evaluated for association with cancer relapse and survival among a cohort of approximately 1,100 breast cancer patients who had been followed for cancer recurrence and mortality for a median of 7.1 years. The T allele was associated with reduced cancer relapse and greater survival, particularly among patients with earlier stage cancer. Among patients of tumor-node-metastasis stage 0 to II, the adjusted hazard ratio of disease-free survival was 0.7 [95% confidence interval (95% CI), 0.5-0.9] for patients carrying T allele compared with those homozygous for the C allele (P = 0.02). In vitro experiments showed that the T allele had higher promoter activity than the C allele in breast cancer cells. Electrophoretic mobility shift assays showed binding of nuclear proteins to the DNA sequence at the SNP site of the T allele but not that of the C allele. The data suggest that MMP8 gene variation may influence breast cancer prognosis and support the notion that MMP8 has an inhibitory effect on cancer metastasis.

Association Between the Chromosome 9p21 Locus and Angiographic Coronary Artery Disease Burden: A Collaborative Meta-Analysis

OBJECTIVES This study sought to ascertain the relationship of 9p21 locus with: 1) angiographic coronary artery disease (CAD) burden; and 2) myocardial infarction (MI) in individuals with underlying CAD. BACKGROUND Chromosome 9p21 variants have been robustly associated with coronary heart disease, but questions remain on the mechanism of risk, specifically whether the locus contributes to coronary atheroma burden or plaque instability. 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 are 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 with angiographically defined cases and control subjects (pooled odds ratio [OR]: 1.31, 95% confidence interval [CI]: 1.20 to 1.43). Among subjects with angiographic CAD (n = 20,987), random-effects model identified an association with multivessel CAD, compared with those with single-vessel disease (OR: 1.10, 95% CI: 1.04 to 1.17)/copy of risk allele). Genotypic models showed an OR of 1.15, 95% CI: 1.04 to 1.26 for heterozygous carrier and OR: 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 control subjects (n = 15,882) had underlying CAD (OR: 0.99, 95% CI: 0.95 to 1.03)/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.

Association of variation at the ABO locus with circulating levels of soluble intercellular adhesion molecule-1, soluble P-selectin, and soluble E-selectin: a meta-analysis.

Background— Circulating levels of soluble intercellular adhesion molecule-1, soluble P-selectin, and soluble E-selectin have been associated with variation at the ABO locus. To evaluate these associations and the effect sizes, we performed a meta-analysis with new and previous reported data for polymorphism rs579459. Methods and Results— Compared with major allele homozygotes, heterozygotes and minor allele homozygotes had 4.6% (95% CI, 3.4%–5.8%, P=7.3×10−14) and 7.2% (95% CI, 4.7%–9.7%, P=1.5×10−8), respectively, lower soluble intercellular adhesion molecule-1 levels (n=33 671). An allele dose-dependent association also was observed for soluble P-selectin (n=4921) with heterozygotes and minor allele homozygotes having 11.5% (95% CI, 7.2%–15.8%, P=1.7×10−7) and 18.6% (95% CI, 9.1%–28.1%, P=1.2×10−4), respectively, lower levels than in major allele homozygotes. A larger effect size, again consistent with an additive genetic model, was seen for soluble E-selectin (n=2860) whose level was 25.6% (95% CI, 19.0%–32.2%, P=2.1×10−14) lower in heterozygotes and 43.3% (95% CI, 36.9%–49.3%, P=4.3×10−42) lower in minor allele homozygotes than in major allele homozygotes. Conclusions— The data support the association of variation at the ABO locus with soluble intercellular adhesion molecule-1, soluble P-selectin, and soluble E-selectin levels.

Association of the lymphotoxin-alpha gene Thr26Asn polymorphism with severity of coronary atherosclerosis.

A recent large-scale, genome-wide association study of single nucleotide polymorphisms showed a strong association between susceptibility to myocardial infarction and the Thr26Asn polymorphism in the lymphotoxin-α (LTA) gene. In the present study, we investigated whether the LTA Thr26Asn polymorphism was associated with the extent of coronary atherosclerosis in a large cohort (n=1082) of well-documented coronary artery disease patients. Thr26Asn genotypes showed a significant different distribution in male patients, when stratified according to the number of diseased coronary arteries, with an odds ratio of 1.98 (95% CI 1.22–3.22) for multiple-vessel disease in patients of the Asn/Asn genotype, compared with patients of the Thr/Thr or Thr/Asn genotype (P=0.006). Thus, further to the recent finding that LTA gene variation is associated with susceptibility to coronary heart disease, the present study provides evidence of an association between LTA genotype and the extent of coronary atherosclerosis.

Coronary Artery Disease–Related Genetic Variant on Chromosome 10q11 Is Associated With Carotid Intima-Media Thickness and Atherosclerosis

Objective—To investigate whether chromosome 10q11.21 influences common carotid intima-media thickness (IMT) and atherosclerosis and whether it is associated with stromal cell-derived factor-1&agr; (SDF-1&agr;) plasma levels. Methods and Results—Variation on chromosome 10q11.21 has been consistently associated with coronary artery disease. The genetic variant lies upstream of the gene encoding SDF-1&agr;. We genotyped 3 population cohorts (Bruneck [age range, 45 to 94 years; 50.0% men; n=738], Health2000 [age range, 46 to 76 years; 55.4% men; n=1237], and essential hypertension in families collected in the region of Oxford [HTO] [age range, 19 to 88 years; 47.9% men; n=770]) for single-nucleotide polymorphism rs501120 at the 10q11.21 locus and conducted a meta-analysis in these cohorts to ascertain a relationship between the polymorphism and carotid IMT. The analysis showed that individuals with the T/T genotype had a significantly higher carotid IMT than individuals with the C/T or C/C genotype (pooled weighted mean difference, 23 &mgr;m [95% CI, 9 to 37 &mgr;m], P=0.0014 under a fixed-effects model; and 23 &mgr;m [95% CI, 6 to 41 &mgr;m], P=0.009 under a random-effects model). In the Bruneck cohort, in which data for carotid atherosclerosis and plasma SDF-1&agr; levels were available, we observed an association of the T/T genotype with a higher burden of atherosclerosis and increased susceptibility to the development of atherosclerosis during a 5-year follow-up (multivariable odds ratio, 1.73 [95% CI, 1.18 to 2.52]; P=0.005 for the recessive model) and an association between the T/T genotype and lower SDF-1&agr; levels (2.62 ng/mL for T/T versus 2.74 ng/mL for C/C or C/T; P=0.023). Conclusion—The coronary heart disease-related variant at the 10q11.21 locus is associated with carotid IMT and atherosclerosis.

ADAM33 expression in atherosclerotic lesions and relationship of ADAM33 gene variation with atherosclerosis.

A-disintegrin-and-metalloproteinase-domains (ADAMs) are membrane-anchored glycoproteins involved in cell adhesion, cell migration and proteolysis. ADAM15 has been implicated in atherosclerosis, with an effect on vascular smooth muscle cell migration. We investigated whether ADAM33, which is evolutionally closely related to ADAM15, was expressed in atheromas and whether it had an effect on vascular smooth muscle migration. We also tested whether ADAM33 gene variation had an influence on the extent of atherosclerosis in patients with coronary artery disease. Immunohistochemical analyses showed that ADAM33 was expressed in smooth muscle cells in the arterial wall and that the expression was increased in smooth muscle cells in atheromas. ADAM33 immunostaining on inflammatory cells in atheromas was also observed. Primary vascular smooth muscle cells in culture were also found to express ADAM33. Boyden chamber assays showed that a neutralising antibody against ADAM33 increased the ability of arterial smooth muscle cells to migrate through a reconstituted basement membrane, suggesting that ADAM33 has an inhibitory effect on vascular smooth muscle migration. Moreover, we detected an association between ADAM33 genotype and the extent of atherosclerosis in a large cohort of coronary artery disease patients. These findings suggest that ADAM33 is implicated in the pathogenesis of atherosclerosis.

Molecular epidemiology of Streptococcus zooepidemicus isolated from the respiratory tracts of Thoroughbred racehorses in training.

The objective of this study was to characterise the molecular epidemiology of Streptococcus zooepidemicus isolated from the respiratory tracts of 198 Thoroughbred racehorses based at three Newmarket training yards over a 10 month period. Typing utilised two separate PCR procedures targeting the M-like protein hypervariable and the 16S-23S RNA gene intergenic spacer regions of the bacterium. S. zooepidemicus, isolated from 23% (224/983) of study samples, comprised 24 different types of varying prevalence. The four most common types, A1HV4, A1HV2, C1HVu and D1HV1, accounted for 45% of all the typed isolates. Overall, the findings were similar to those reported in a study of Welsh Mountain ponies and confirm that in equids, S. zooepidemicus is not a homogeneous, clonal population but instead represents a wide diversity of strain types. This diversity also highlights potential difficulties in using vaccines to control S. zooepidemicus infections in horses as it is likely that the induction of a heterologous, cross-protective immunity will be required.