Anders Hamsten

Fatty acid desaturases in human adipose tissue: relationships between gene expression, desaturation indexes and insulin resistance

Aims/hypothesisFatty acid desaturases introduce double bonds into growing fatty acid chains. The key desaturases in humans are Δ5-desaturase (D5D), Δ6-desaturase (D6D) and stearoyl-CoA desaturase (SCD). Animal and human data implicate hepatic desaturase activities in insulin resistance, obesity and dyslipidaemia. However, the role of desaturase activity in adipose tissue is uncertain. We therefore evaluated relationships between adipose mRNA expression, estimated desaturase activities (fatty acid ratios) in adipose tissue and insulin resistance.MethodsSubcutaneous adipose tissue mRNA expression of D5D (also known as FADS1), D6D (also known as FADS2) and SCD was determined in 75 individuals representative of the study population of 294 healthy 63-year-old men. Desaturation indexes (product/substrate fatty acid ratios) were generated from adipose tissue fatty acid composition in all individuals. Insulin resistance was defined as the upper quartile of the updated homeostasis model assessment (HOMA-2) index.ResultsThe relevant desaturation indexes (16:1/16:0, 18:1/18:0, 20:4/20:3 and 18:3/18:2) reflected expression of SCD, but not of D5D or D6D in adipose tissue. Insulin-resistant individuals had a higher adipose tissue 18:1/18:0, but not 16:1/16:0 ratio than insulin-sensitive individuals. Individuals with a high adipose tissue 18:1/18:0 ratio were 4.4-fold (95% CI 1.8–11.8) more likely to be insulin resistant [threefold (95% CI 1.1–8.6) after adjustment for waist circumference and plasma triacylglycerol]. In a multiple regression model predicting HOMA-2, the independent effect of the 18:1/18:0 ratio was borderline (p = 0.086).Conclusions/interpretationAdipose tissue desaturation indexes of SCD reflect the expression of the gene encoding the enzyme in this tissue. Elevated SCD activity within adipose tissue is closely coupled to the development of insulin resistance.

Serum matrix metalloproteinase-3 concentration is influenced by MMP-3 )1612 5A/6A promoter genotype and associated with myocardial infarction

Objectives.  Matrix metalloproteinase‐3 (MMP‐3) is implicated in the formation of atherosclerotic plaques, and the MMP‐3 −1612 5A/6A polymorphism is associated with myocardial infarction (MI) and stable coronary artery disease (CAD). The present study examined whether the −1612 5A/6A polymorphism in the promoter region of the MMP‐3 gene influences serum concentrations of MMP‐3 and whether serum concentrations of MMP‐3 are related to extent of coronary atherosclerosis and risk of MI.

Measurements of carotid intima-media thickness and of interadventitia common carotid diameter improve prediction of cardiovascular events: results of the IMPROVE (Carotid Intima Media Thickness [IMT] and IMT-Progression as Predictors of Vascular Events in a High Risk European Population) study.

OBJECTIVES The goal of this study was to compare the performance of several measures of carotid intima-media thickness (C-IMT) as predictors of cardiovascular events (CVEs), and to investigate whether they add to the predictive accuracy of Framingham risk factors (FRFs). BACKGROUND Various markers of subclinical atherosclerosis have been identified as predictors of CVEs, but the most powerful variable is still under debate. METHODS A cohort study was carried out in 5 European countries. A total of 3,703 subjects (median age 64.4 years; 48% men) were followed-up for a median of 36.2 months, and 215 suffered a first CVE (incidence: 19.9/1,000 person-years). RESULTS All measures of C-IMT and the interadventitia common carotid artery diameter (ICCAD) were associated with the risk of CVEs, after adjustment for FRFs and therapies (all p < 0.005). The average of 8 maximal IMT measurements (IMT(mean-max)), alone or combined with ICCAD, classified events and non-events better than the common carotid mean IMT (net reclassification improvement [NRI]: +11.6% and +19.9%, respectively; both p < 0.01). Compared with classification based on FRFs alone, the NRI resulting from the combination of FRFs+ICCAD+IMT(mean-max) was +12.1% (p < 0.01). The presence of at least 1 plaque (maximum IMT >1.5 mm) performed significantly worse than composite IMTs that incorporated plaques (p < 0.001). Adjusted Kaplan-Meier curves showed that individuals with a FRS = 22.6% (cohort average), and both IMT(mean-max) and ICCAD above the median, had a 6.5% risk to develop a CVE over 3 years versus a 3.4% risk for those with the same FRS, and both IMT(mean-max) and ICCAD below the median. CONCLUSIONS A risk stratification strategy based on C-IMT and ICCAD as an adjunct to FRFs is a rational approach to prevention of cardiovascular disease.

Association of Genetic Risk Variants With Expression of Proximal Genes Identifies Novel Susceptibility Genes for Cardiovascular Disease

Background—Population-based genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with cardiovascular disease or its risk factors. Genes in close proximity to these risk-SNPs are often thought to be pathogenetically important based on their location alone. However, the actual connections between SNPs and disease mechanisms remain largely unknown. Methods and Results—To identify novel susceptibility genes, we investigated how 166 SNPs previously found to be associated with increased cardiovascular risk and/or predisposing metabolic traits relate to the expression of nearby genes. Gene expression in 577 samples of aorta, liver, mammary artery, and carotid atherosclerotic plaque was measured using expression arrays. For 47 SNPs, the expression levels of proximal genes (located within 200 kb) were affected (P<0.005). More than 20 of these genes had not previously been identified as candidate genes for cardiovascular or related metabolic traits. SNP-associated gene effects were tissue-specific and the tissue specificity was phenotype-dependent. Conclusions—This study demonstrates several instances of association between risk-SNPs and genes immediately adjacent to them. It also demonstrates instances in which the associated gene is not the immediately proximal and obvious candidate gene for disease. This shows the necessity of careful studies of genetic marker data as a first step toward application of genome-wide association studies findings in a clinical setting.

Concentration of apolipoprotein B is comparable with the apolipoprotein B/apolipoprotein A-I ratio and better than routine clinical lipid measurements in predicting coronary heart disease mortality: findings from a multi-ethnic US population

AIMS Prospective studies indicate that apolipoprotein measurements predict coronary heart disease (CHD) risk; however, evidence is conflicting, especially in the US. Our aim was to assess whether measurements of apolipoprotein B (apoB) and apolipoprotein A-I (apoA-I) can improve the ability to predict CHD death beyond what is possible based on traditional cardiovascular (CV) risk factors and clinical routine lipid measurements. METHODS AND RESULTS We analysed prospectively associations of apolipoprotein measurements, traditional CV risk factors, and clinical routine lipid measurements with CHD mortality in a multi-ethnic representative subset of 7594 US adults (mean age 45 years; 3881 men and 3713 women, median follow-up 124 person-months) from the Third National Health and Nutrition Examination Survey mortality study. Multiple Cox-proportional hazards regression was applied. There were 673 CV deaths of which 432 were from CHD. Concentrations of apoB [hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.09-3.61], apoA-I (HR 0.48, 95% CI 0.27-0.85) and total cholesterol (TC) (HR 1.17, 95% CI 1.02-1.34) were significantly related to CHD death, whereas high density lipoprotein cholesterol (HDL-C) (HR 0.68, 95% CI 0.45-1.05) was borderline significant. Both the apoB/apoA-I ratio (HR 2.14, 95% CI 1.11-4.10) and the TC/HDL-C ratio (HR 1.10, 95% CI 1.04-1.16) were related to CHD death. Only apoB (HR 2.01, 95% CI 1.05-3.86) and the apoB/apoA-I ratio (HR 2.09, 95% CI 1.04-4.19) remained significantly associated with CHD death after adjusting for CV risk factors. CONCLUSION In the US population, apolipoprotein measurements significantly predict CHD death, independently of conventional lipids and other CV risk factors (smoking, dyslipidaemia, hypertension, obesity, diabetes and C-reactive protein). Furthermore, the predictive ability of apoB alone to detect CHD death was better than any of the routine clinical lipid measurements. Inclusion of apolipoprotein measurements in future clinical guidelines should not be discarded.

Transcriptional profiling uncovers a network of cholesterol-responsive atherosclerosis target genes.

Despite the well-documented effects of plasma lipid lowering regimes halting atherosclerosis lesion development and reducing morbidity and mortality of coronary artery disease and stroke, the transcriptional response in the atherosclerotic lesion mediating these beneficial effects has not yet been carefully investigated. We performed transcriptional profiling at 10-week intervals in atherosclerosis-prone mice with human-like hypercholesterolemia and a genetic switch to lower plasma lipoproteins (Ldlr −/− Apo 100/100 Mttp flox/flox Mx1-Cre). Atherosclerotic lesions progressed slowly at first, then expanded rapidly, and plateaued after advanced lesions formed. Analysis of lesion expression profiles indicated that accumulation of lipid-poor macrophages reached a point that led to the rapid expansion phase with accelerated foam-cell formation and inflammation, an interpretation supported by lesion histology. Genetic lowering of plasma cholesterol (e.g., lipoproteins) at this point all together prevented the formation of advanced plaques and parallel transcriptional profiling of the atherosclerotic arterial wall identified 37 cholesterol-responsive genes mediating this effect. Validation by siRNA-inhibition in macrophages incubated with acetylated-LDL revealed a network of eight cholesterol-responsive atherosclerosis genes regulating cholesterol-ester accumulation. Taken together, we have identified a network of atherosclerosis genes that in response to plasma cholesterol-lowering prevents the formation of advanced plaques. This network should be of interest for the development of novel atherosclerosis therapies.

Genome-wide association study for circulating levels of PAI-1 provides novel insights into its regulation.

We conducted a genome-wide association study to identify novel associations between genetic variants and circulating plasminogen activator inhibitor-1 (PAI-1) concentration, and examined functional implications of variants and genes that were discovered. A discovery meta-analysis was performed in 19 599 subjects, followed by replication analysis of genome-wide significant (P < 5 × 10(-8)) single nucleotide polymorphisms (SNPs) in 10 796 independent samples. We further examined associations with type 2 diabetes and coronary artery disease, assessed the functional significance of the SNPs for gene expression in human tissues, and conducted RNA-silencing experiments for one novel association. We confirmed the association of the 4G/5G proxy SNP rs2227631 in the promoter region of SERPINE1 (7q22.1) and discovered genome-wide significant associations at 3 additional loci: chromosome 7q22.1 close to SERPINE1 (rs6976053, discovery P = 3.4 × 10(-10)); chromosome 11p15.2 within ARNTL (rs6486122, discovery P = 3.0 × 10(-8)); and chromosome 3p25.2 within PPARG (rs11128603, discovery P = 2.9 × 10(-8)). Replication was achieved for the 7q22.1 and 11p15.2 loci. There was nominal association with type 2 diabetes and coronary artery disease at ARNTL (P < .05). Functional studies identified MUC3 as a candidate gene for the second association signal on 7q22.1. In summary, SNPs in SERPINE1 and ARNTL and an SNP associated with the expression of MUC3 were robustly associated with circulating levels of PAI-1.

Identifying the susceptibility genes for coronary artery disease: from hyperbole through doubt to cautious optimism

The genetic basis of coronary artery disease (CAD) is complex, and the fact that an alarmingly high proportion of reported associations between genetic variants and CAD are not replicated has generated uncertainty as to whether molecular genetics is ever going to deliver on the promises delivered in the late 1990s. However, during 2007, the first generation of large‐scale genome‐wide association studies using high‐density, single nucleotide polymorphism genotyping arrays have revealed genetic variants that are robustly associated with CAD and CAD‐related traits such as type 2 diabetes and obesity. In particular, a robust susceptibility locus for CAD has been identified on chromosome 9p21. Also, evidence has been obtained that multiple rare alleles with fairly strong phenotypic effects may contribute to the genetic heritability of CAD, in addition to common variants with a modest impact on risk. Furthermore, new mechanistic connections have been discovered between different common complex diseases including CAD. This review focuses on the challenges and recent advances of molecular genetics in dissecting the molecular pathophysiology of atherothrombosis and defining novel targets for treatment.

IGF-I/IGFBP-3 ratio: a mechanistic insight into the metabolic syndrome

Recent reports suggest that IGF (insulin-like growth factor)-I and IGFBP-3 (IGF-binding protein-3) have independent and opposing mechanistic effects on insulin. The aim of the present study was to assess the relationship between the IGF-I/IGFBP-3 ratio and the metabolic syndrome. We examined 3281 subjects (1463 men and 1818 women, aged 20-49 years), otherwise healthy adults, who participated in NHANES III (Third National Health and Nutrition Examination Survey), which has released measurements of IGF-I and IGFBP-3. Insulin resistance was estimated using the computer HOMA2 (homoeostatic model assessment 2) model. The updated ATP-III (Adult Treatment Panel III) definition of the metabolic syndrome was used. We applied adjusted logistic and linear regression models. After adjusting for age and race, men and women in the lowest quartile of the IGF-I/IGFBP-3 ratio were 3-fold more likely to meet the ATP-III definition of the metabolic syndrome and twice as likely to be insulin-resistant. Mean values of the IGF-I/IGFBP-3 ratio decreased significantly as the number of metabolic syndrome components increased (P<0.0001, as determined by ANOVA). The area under the ROC (receiver operating characteristic) curve for detecting insulin resistance using the IGF-I/IGFBP-3 ratio was 0.760, significantly improving upon either protein alone (P=0.01). In conclusion, the IGF-I/IGFBP-3 ratio is significantly associated with the metabolic syndrome. Calculating the ratio of these two proteins may provide insight into the metabolic syndrome clustering phenomenon.

Oxidised‐HDL3 induces the expression of PAI‐1 in human endothelial cells. Role of p38MAPK activation and mRNA stabilization

Modified lipoproteins have been suggested to modulate endothelial expression of plasminogen activator inhibitor‐1 (PAI‐1). As oxidized high‐density lipoprotein (Ox‐HDL) has been found in atheromatous plaques and receptors for modified HDL are present on endothelial cells, we investigated the role of Ox‐HDL3 on the expression of PAI‐1. Ox‐HDL3 but not native HDL3, increased PAI‐1 mRNA expression in endothelial cells. Furthermore, PAI‐1 antigen expression and activity increased in the supernatant of cells incubated with Ox‐HDL3. The intracellular pathways involved in this effect were investigated. Ox‐HDL3 activated both extracellular signal‐regulated kinases (ERK) 1/2 and p38 mitogen‐activated protein kinase (MAPK). Moreover, incubation with specific inhibitors of these kinases showed that p38MAPK was mainly involved in the Ox‐HDL3‐dependent PAI‐1 induction. Transient transfection experiments suggested that none of the response elements in the proximal promoter (−804 to 17) were involved in Ox‐HDL3‐mediated PAI‐1 expression. mRNA stability experiments showed that Ox‐HDL3 increased the PAI‐1 mRNA half‐life. In summary, Ox‐HDL3 induced PAI‐1 mRNA expression and antigen release through a molecular mechanism involving MAPK activation and mRNA stabilization. Thus, oxidative modification converts HDL to a prothrombotic lipoprotein species.