Salzburg Atherosclerosis Prevention Program In Subjects At High Individual Risk Research Articles

Higher spermidine intake is linked to lower mortality: a prospective population-based study

Spermidine administration is linked to increased survival in several animal models. The aim of this study was to test the potential association between spermidine content in diet and mortality in humans. This prospective community-based cohort study included 829 participants aged 45-84 y, 49.9% of whom were male. Diet was assessed by repeated dietitian-administered validated food-frequency questionnaires (2540 assessments) in 1995, 2000, 2005, and 2010. During follow-up between 1995 and 2015, 341 deaths occurred. All-cause mortality (deaths per 1000 person-years) decreased across thirds of increasing spermidine intake from 40.5 (95% CI: 36.1, 44.7) to 23.7 (95% CI: 20.0, 27.0) and 15.1 (95% CI: 12.6, 17.8), corresponding to an age-, sex- and caloric intake-adjusted 20-y cumulative mortality incidence of 0.48 (95% CI: 0.45, 0.51), 0.41 (95% CI: 0.38, 0.45), and 0.38 (95% CI: 0.34, 0.41), respectively. The age-, sex- and caloric ratio-adjusted HR for all-cause death per 1-SD higher spermidine intake was 0.74 (95% CI: 0.66, 0.83; P<0.001). Further adjustment for lifestyle factors, established predictors of mortality, and other dietary features yielded an HR of 0.76 (95% CI: 0.67, 0.86; P<0.001). The association was consistent in subgroups, robust against unmeasured confounding, and independently validated in the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) Study (age-, sex-, and caloric ratio-adjusted HR per 1-SD higher spermidine intake: 0.71; 95% CI: 0.53, 0.95; P=0.019). The difference in mortality risk between the top and bottom third of spermidine intakes was similar to that associated with a 5.7-y (95% CI: 3.6, 8.1 y) younger age. Our findings lend epidemiologic support to the concept that nutrition rich in spermidine is linked to increased survival in humans. This trial was registered at www.clinicaltrials.gov as NCT03378843.

Association Between Vascular Cell Adhesion Molecule 1 and Atrial Fibrillation

Accumulating evidence links inflammation and atrial fibrillation (AF). To assess whether markers of systemic and atrial inflammation are associated with incident AF in the general population. The Bruneck Study is a prospective, population-based cohort study with a 20-year follow-up (n = 909). The population included a random sample of the general community aged 40 to 79 years. Levels of 13 inflammation markers were measured at baseline in 1990. Findings were replicated in a case-control sample nested within the prospective Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study (n = 1770). Data analysis was performed from February to May 2016. Levels of 13 inflammation markers. Incident AF over a 20-year follow-up period in the Bruneck Study. Of the 909 participants included in the Bruneck Study, mean [SD] age was 58.8 (11.4) years and 448 (49.3%) were women. Among the 880 participants free of prevalent AF (n = 29) at baseline, 117 developed AF during the 20-year follow-up period (incidence rate, 8.2; 95% CI, 6.8-9.6 per 1000 person-years). The levels of soluble vascular cell adhesion molecule 1 (VCAM-1) and osteoprotegerin were significantly associated with incident AF (hazard ratio [HR], 1.49; 95% CI, 1.26-1.78; and 1.46; 95% CI, 1.25-1.69, respectively; P < .001 with Bonferroni correction for both), but osteoprotegerin lost significance after age and sex adjustment (HR, 1.05; 95% CI, 0.87-1.27; P > .99 with Bonferroni correction). Matrix metalloproteinase 9, metalloproteinase inhibitor 1, monocyte chemoattractant protein-1, P-selectin, fibrinogen, receptor activator of nuclear factor-κB ligand, high-sensitivity C-reactive protein, adiponectin, leptin, soluble intercellular adhesion molecule 1, and E-selectin all fell short of significance (after Bonferroni correction in unadjusted and age- and sex-adjusted analyses). The HR for a 1-SD higher soluble VCAM-1 level was 1.34 (95% CI, 1.11-1.62; Bonferroni-corrected P = .03) in a multivariable model. The association was of a dose-response type, at least as strong as that obtained for N-terminal pro-B-type natriuretic peptide (multivariable HR for a 1-SD higher N-terminal pro-B-type natriuretic peptide level, 1.15; 95% CI, 1.04-1.26), internally consistent in various subgroups, and successfully replicated in the SAPHIR Study (age- and sex-adjusted, and multivariable odds ratios for a 1-SD higher soluble VCAM-1 level, 1.91; 95% CI, 1.24-2.96, P = .003; and 2.59; 95% CI, 1.45-4.60; P = .001). Levels of soluble VCAM-1, but not other inflammation markers, are significantly associated with new-onset AF in the general community. Future studies should address whether soluble VCAM-1 is capable of improving AF risk classification beyond the information provided by standard risk scores.

Body adiposity index and other indexes of body composition in the SAPHIR study: Association with cardiovascular risk factors

The accuracy of anthropometric surrogate markers such as the body adiposity index (BAI) and other common indexes like the body mass index (BMI), waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) to predict metabolic sequelae is essential for its use in clinical practice. Thus, we evaluated the strength of BAI and other indexes to relate with anthropometric parameters, adipocytokines, blood lipids, parameters of glucose-homeostasis and blood pressure in 1,770 patients from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study in a crosssectional design. Measurements were BAI, BMI, WHR, WHtR, abdominal subcutaneous and visceral adipose tissue (aSAT and VAT), total body adipose tissue mass, body weight, waist- and hip circumference (WC and HC), leptin, adiponectin, high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), triglycerides (TG), fasting plasma glucose, fasting plasma insulin, the homeostasis model assessment of insulin resistance (HOMAIR), systolic and diastolic blood pressure. BAI was significantly associated with leptin and HC. We conclude that BAI was the best calculator for leptin. BAI was inferior to BMI to predict anthropometric parameters other than HC, adiponectin, blood lipids, parameters of glucose homeostasis, and blood pressure in this cross-sectional study.

Cholesteryl ester transfer protein and hepatic lipase gene polymorphisms: Effects on hepatic mRNA levels, plasma lipids and carotid atherosclerosis

ObjectiveHDL modifying effects of cholesteryl ester transfer protein (CETP) and hepatic lipase (LIPC) depend in part on each other. We studied associations of CETP-Taq1B and -514C>T-LIPC polymorphisms with hepatic mRNA levels, and their combined effects on plasma lipids and carotid atherosclerosis. MethodsWe genotyped the CETP-Taq1B and the -514C>T-LIPC polymorphisms in 67 obese women in whom hepatic CETP and LIPC transcript levels were determined as well as in 1549 participants of the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR). Carotid atherosclerosis was assessed by intima-media thickness and extent of plaques (B-score) of the carotid arteries. ResultsIn obese women, CETP-Taq1B and -514C>T-LIPC variant alleles were associated with reduced hepatic levels of CETP and LIPC mRNA, respectively. The CETP and LIPC polymorphisms accounted for 12.9 and 14.4% of the variability in respective transcripts. In the SAPHIR population, CETP-Taq1B showed independent effects on LDL diameter, HDL and LDL cholesterol, apolipoproteins AI and B and cholesterol/HDL cholesterol, while -514C>T-LIPC revealed independent effects on HDL cholesterol and apolipoprotein AI. The two polymorphisms displayed interactions at the level of HDL cholesterol. Compared to subjects carrying wild-type alleles at both loci, subjects homozygous for the CETP wild-type allele, but heterozygous for the LIPC polymorphism and subjects heterozygous for the CETP polymorphism, but homozygous for the LIPC wild-type allele showed an increased risk of carotid atherosclerosis (both P<0.05). ConclusionsCETP and LIPC polymorphisms influence the respective hepatic transcript levels, demonstrate interactions on HDL cholesterol and suggest that imbalances between CETP and LIPC activities may modulate the risk of carotid atherosclerosis.

Investigation and Functional Characterization of Rare Genetic Variants in the Adipose Triglyceride Lipase in a Large Healthy Working Population

Recent studies demonstrated a strong influence of rare genetic variants on several lipid-related traits. However, their impact on free fatty acid (FFA) plasma concentrations, as well as the role of rare variants in a general population, has not yet been thoroughly addressed. The adipose triglyceride lipase (ATGL) is encoded by the PNPLA2 gene and catalyzes the rate-limiting step of lipolysis. It represents a prominent candidate gene affecting FFA concentrations. We therefore screened the full genomic region of ATGL for mutations in 1,473 randomly selected individuals from the SAPHIR (Salzburg Atherosclerosis Prevention program in subjects at High Individual Risk) Study using a combined Ecotilling and sequencing approach and functionally investigated all detected protein variants by in-vitro studies. We observed 55 novel mostly rare genetic variants in this general population sample. Biochemical evaluation of all non-synonymous variants demonstrated the presence of several mutated but mostly still functional ATGL alleles with largely varying residual lipolytic activity. About one-quarter (3 out of 13) of the investigated variants presented a marked decrease or total loss of catalytic function. Genetic association studies using both continuous and dichotomous approaches showed a shift towards lower plasma FFA concentrations for rare variant carriers and an accumulation of variants in the lower 10%-quantile of the FFA distribution. However, the generally rather small effects suggest either only a secondary role of rare ATGL variants on the FFA levels in the SAPHIR population or a recessive action of ATGL variants. In contrast to these rather small effects, we describe here also the first patient with “neutral lipid storage disease with myopathy” (NLSDM) with a point mutation in the catalytic dyad, but otherwise intact protein.

Are plasma VEGF and its soluble receptor sFlt-1 atherogenic risk factors? Cross-sectional data from the SAPHIR study

AimsVascular endothelial growth factor (VEGF) is a potent hypoxia-regulated angiogenic factor. Its soluble receptor soluble (s)Flt-1 binds VEGF with high affinity inhibiting the angiogenic function of VEGF. The role of circulating VEGF in atherosclerosis is unclear. Methods and resultsIn 909 healthy subjects (511 male, 398 female) from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) we determined fasting plasma VEGF and sFlt-1 concentration, cardiovascular risk factors and carotid atherosclerosis. VEGF levels were lower and sFlt-1 levels higher in men than in women. VEGF and sFlt-1 showed a positive correlation. In the entire population VEGF correlated positively with age, BMI, insulin resistance, white blood cell and platelet count, C-reactive protein (CRP) and carotid intima media thickness (IMT). After adjustment for age, VEGF showed a weak positive correlation with BMI, liver enzymes, CRP and platelet count in males. In females VEGF correlated negatively with LDL-cholesterol and positively with insulin resistance and platelet count. After adjustment for age, no significant correlation with carotid atherosclerosis could be detected. ConclusionPlasma VEGF and sFlt-1 are only weakly correlated with cardiovascular risk factors, suggesting that circulating VEGF levels do have only a minor impact on the development of atherosclerosis.

Effect of obesity and insulin sensitivity on adiponectin isoform distribution

Adiponectin is an insulin-sensitizing, antiatherogenic and anti-inflammatory adipocytokine that circulates in three isoforms: a trimer [low-molecular weight (LMW)], a hexamer (trimer-dimer) of medium molecular weight (MMW) and a multimeric high molecular weight (HMW) isoform. Evidence is accumulating that HMW adiponectin is the active isoform of the adipocytokine. We investigated the impact of adipose tissue and insulin sensitivity on adiponectin isoform distribution. One hundred and eighty-seven normolipidaemic, non-diabetic lean or obese subjects with or without insulin resistance participating in the Salzburg Atherosclerosis Prevention program in subjects at High Individual Risk (SAPHIR) were included in the study. Insulin sensitivity was determined by the short insulin tolerance test and the homeostasis model assessment (HOMA) index. Serum adiponectin isoform distribution was determined by an enzyme immunoassay. Total adiponectin as well as HMW/total adiponectin ratio was significantly increased in female subjects. Circulating total adiponectin levels were lowest in obese patients due to reduced concentrations of HMW adiponectin. As determined by stepwise regression analysis, besides age and high density lipoprotein (HDL) cholesterol, visceral fat area and waist-to-hip ratio predicted concentrations of HMW adiponectin, while insulin sensitivity had no influence on either total adiponectin or its isoforms. Our results underline that determination of adiponectin isoforms are more useful than measurement of total adiponectin in clinical settings. Our data suggest that adiponectin concentrations are strongly associated with visceral fat area but not with insulin sensitivity. Thus, we hypothesize that insulin resistance is a consequence rather than the cause of hypoadiponectinaemia in obese subjects.

INSIG2 Polymorphism Is Neither Associated With BMI Nor With Phenotypes of Lipoprotein Metabolism

A previous epidemiological study showed an association of the insulin-induced gene 2 (INSIG2) gene with BMI. Additionally, experimental investigations in animals and cell culture provided evidence that this gene might be involved in lipoprotein and free fatty acid (FFA) metabolism. Therefore, the aim of this study was to examine the association between the rs7566605 variant near the INSIG2 gene and BMI and to extend it to other quantitative measures of obesity, as well as parameters of lipoprotein and FFA metabolism. We genotyped rs7566605 in a group of severely obese white patients (n = 1,026) with an average BMI of 46.0 kg/m(2) and a control group (n = 818) from Utah, as well as in the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study from Austria, which is based on a healthy working population (n = 1,696). We observed no difference in the genotype frequency of rs7566605 of INSIG2 between obese subjects and population-based controls from Utah. Furthermore, we did not find evidence of an association with measures of body composition (BMI, waist, waist-to-hip ratio, percentage body fat, amount of visceral and subcutaneous abdominal adipose fat) or lipoprotein metabolism (total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, triglycerides, and FFAs) in the Utah study population or in the independent SAPHIR study. Our results do not support an association of the INSIG2 gene with the regulation of body weight or parameters related to lipoprotein metabolism.

APOA5 variants and metabolic syndrome in Caucasians

Apolipoprotein A5 (APOA5) gene variants were reported to be associated with two components of metabolic syndrome (MetS): higher TG levels and lower HDL levels. Moreover, a recent Japanese case-control study found variant -1131T>C associated with MetS itself. Thus, our study systematically analyzed the APOA5 gene for association with lipid parameters, any other features of MetS, including waist circumference, glucose-related parameters, blood pressure, uric acid, and MetS itself in Caucasians. Ten polymorphisms were analyzed in a large fasting sample of the population-based Cooperative Health Research in the Region of Augsburg (KORA) survey S4 (n = 1,354; southern Germany) and in a second fasting sample, the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study (n = 1,770; Austria). Minor alleles of variants -1131T>C, -3A>G, c.56C>G, 476G>A, and 1259T>C were significantly associated with higher TG levels in single polymorphism (P < 0.001) and haplotype (P <or= 6.6 x 10(-6)) analysis. Besides associations with lower HDL levels in SAPHIR (P <or= 0.001), there were no significant findings with any other features of MetS. Variant c.56C>G was associated with higher risk for MetS [odds ratio (95% confidence interval) = 1.43 (1.04, 1.99), P = 0.03 for KORA and 1.48 (1.10, 1.99), P = 0.009 for SAPHIR). Our study confirms the association of the APOA5 locus with TG and HDL levels in humans. Furthermore, the data suggest a different mechanism of APOA5 impact on MetS in Caucasians, as variant c.56C>G (not analyzed in the Japanese study) and not -1131T>C, as in the Japanese subjects, was associated with MetS.

Gender-specific effect of metabolic syndrome on rate adjusted QT interval in middle-aged participants of an atherosclerosis prevention program

The metabolic syndrome is a matter of immense public concern for atherosclerosis prevention. Key features are visceral obesity, dyslipidemia, hyperglycemia in the non-diabetic range, and arterial hypertension. Subclinical atherosclerosis is the clinical consequence of metabolic syndrome, which may influence the QT interval. The aim was to investigate the rate corrected QT interval in subjects with metabolic syndrome in comparison to those without cardiometabolic risk factor clusters, and to explore gender differences in cardiac repolarization between the two groups. Heart rate and QT interval were automatically measured from surface ECG in 1086 participants (767 men, 319 women) from the Salzburg-Atherosclerosis-Prevention-program-in-subjects-at-High-Individual-Risk (SAPHIR). To omit the QT adjustment bias inherent in Bazett's formula we used a QT adjustment method with linear scaling as described by Rautaharju. The prevalence of metabolic syndrome was 13.8% among males and 10% among females. Mean rate adjusted QT (QTa) intervals were longer in women than in men. Presence of metabolic syndrome, however, was associated with significantly prolonged QTa only in men but not in women. Adjustment for relevant confounders reduced the difference of mean QTa in men from 9.24 to 5.83 ms (95% CI 0.9-10.8), but this difference was still statistically significant (p = 0.021). The effect of metabolic syndrome on QTa was only partly mediated by hypertension and insulin resistance. In females, however, no relevant differences were detected for QTa interval between subjects categorized by presence or absence of metabolic syndrome. The findings indicate a significant association between metabolic syndrome and rate-invariant QT in middle-aged men after adjustment for other risk factors. QT measurement may provide additive diagnostic and prognostic information in populations undergoing cardiovascular risk screening. However, the effect of metabolic and hormonal factors on ventricular repolarization seems to differ between the sexes.

Comparison of different definitions of the metabolic syndrome

The International Diabetes Federation (IDF) published a new definition of the metabolic syndrome (MetS). For this definition we compared frequency, concordance, clinical and laboratory stigmata and carotid atherosclerosis with those of the established definitions by the National Cholesterol Education Program (NCEP) and World Health Organization (WHO). A total of 1518 subjects (943 men, 575 women) from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR), free of clinical atherosclerosis, were included in this study. To estimate insulin sensitivity two methods, i.e. homeostasis model assessment of insulin resistance (HOMA-IR) and the short insulin tolerance test, were employed. Carotid intima media thickness (IMT) and plaque extent were quantified for all subjects using high-resolution ultrasound. Prevalence of the MetS was 18.7% for men and 16.2% for women for the WHO definition, 18.9% and 17.0%, respectively, for the NCEP definition, and 25.8% and 19.5%, respectively, for the IDF definition. Concordance was lower between the definitions of WHO and IDF (< 50%) than between NCEP and IDF (> 67%). Compared to subjects identified by NCEP definition, subjects identified in excess by IDF (3.1-11.7%) showed less insulin resistance and lower IMT and plaque extent indistinguishable from MetS-free subjects. Our data suggest that the IDF definition includes subjects as MetS sufferers above these detected by NCEP or WHO, who exhibit considerably less insulin resistance and carotid atherosclerosis blurring the distinction between health and disease.

Cholesteryl Ester Transfer Protein in Metabolic Syndrome

Low high-density lipoprotein cholesterol (HDL-C), hypertriglyceridemia, and small dense-low density lipoprotein (LDL) are key components of metabolic syndrome (MS). Cholesteryl ester transfer protein (CETP) mediates the transfer of triglycerides (TGs) from TG-rich lipoproteins to HDL and LDL particles in exchange for cholesteryl esters, leading to low HDL-C and small dense-LDL. The aim of this study was to investigate the role of CETP in subjects with MS. In a cross-sectional cohort of 234 middle-aged men and 252 women randomly selected from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study, MS was diagnosed according to the National Cholesterol Education Program guidelines. CETP mass was determined by enzyme-linked immunosorbent assay and LDL size-by-gradient polyacrylamide gel electrophoresis. Men and women with MS had lower HDL-C (45 +/- 7 vs. 58 +/- 13 and 48 +/- 10 vs. 71 +/- 14 mg/dL for men and women, respectively; p < 0.001 for all) and higher TG levels (222 +/- 71 vs. 98 +/- 54 and 167 +/- 67 vs. 90 +/- 35 mg/dL for men and women, respectively; p < 0.001 for all) than healthy subjects. LDL size was lower in subjects with MS (256 +/- 11 A vs. 267 +/- 11 A and 262 +/- 10 A vs. 273 +/- 8 A for men and women, respectively; p < 0.001 for all). CETP mass was higher in men with MS (1.87 +/- 0.78 vs. 1.40 +/- 0.65 mug/mL; p < 0.001) but not in women (1.74 +/- 0.79 vs. 1.62 +/- 0.62 mug/mL). CETP mass correlated inversely with LDL size in both men and women (r = -0.19, p < 0.01 and r = -0.13, p < 0.05 in men and women, respectively). MS is associated with increased CETP mass in men. Increased CETP mass may be responsible for reduced HDL-C and reduced LDL particle diameter in MS.

Associations of the UCP2 Gene Locus With Asymptomatic Carotid Atherosclerosis in Middle-Aged Women

Reactive oxygen species (ROS) contribute to atherogenesis. Uncoupling protein 2 (UCP2) reduces mitochondrial ROS generation and protects against the disease in animal models. A common -866G/A promoter polymorphism that has been associated with obesity and beta-cell function may also affect UCP2 gene expression in cells of the arterial wall. Genotype distributions of the -866G/A and of a 45nt-del/ins polymorphism in the 3'-untranslated region of the UCP2 gene were determined in 1334 participants of the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR). We observed a modest association of the -866G/A promoter polymorphism and 2-loci haplotypes with asymptomatic carotid atherosclerosis in female study participants. Functional studies revealed increased expression of the -866G wild-type allele in human umbilical vein endothelial cells and differentiated THP-1 cells. Electrophoretic mobility shift assay studies and antibody-interference assays performed with nuclear extracts of various cell lines showed binding of cell-type specific protein complexes to the region encompassing the -866 site and suggested involvement of hypoxia inducible factor 1alpha in the regulation of UCP2 gene expression in endothelial cells and macrophages. Our results suggest a role of UCP2 in atherogenesis as originally proposed from studies in animal and cell culture models.