Lowering of Circulating Sclerostin May Increase Risk of Atherosclerosis and Its Risk Factors: Evidence From a Genome-Wide Association Meta-Analysis Followed by Mendelian Randomization.

OBJECTIVE: In this study, we aimed to establish the causal effects of lowering sclerostin, target of the antiosteoporosis drug romosozumab, on atherosclerosis and its risk factors. METHODS: A genome-wide association study meta-analysis was performed of circulating sclerostin levels in 33,961 European individuals. Mendelian randomization (MR) was used to predict the causal effects of sclerostin lowering on 15 atherosclerosis-related diseases and risk factors. RESULTS: We found that 18 conditionally independent variants were associated with circulating sclerostin. Of these, 1 cis signal in SOST and 3 trans signals in B4GALNT3, RIN3, and SERPINA1 regions showed directionally opposite signals for sclerostin levels and estimated bone mineral density. Variants with these 4 regions were selected as genetic instruments. MR using 5 correlated cis-SNPs suggested that lower sclerostin increased the risk of type 2 diabetes mellitus (DM) (odds ratio [OR] 1.32 [95% confidence interval (95% CI) 1.03-1.69]) and myocardial infarction (MI) (OR 1.35 [95% CI 1.01-1.79]); sclerostin lowering was also suggested to increase the extent of coronary artery calcification (CAC) (ß = 0.24 [95% CI 0.02-0.45]). MR using both cis and trans instruments suggested that lower sclerostin increased hypertension risk (OR 1.09 [95% CI 1.04-1.15]), but otherwise had attenuated effects. CONCLUSION: This study provides genetic evidence to suggest that lower levels of sclerostin may increase the risk of hypertension, type 2 DM, MI, and the extent of CAC. Taken together, these findings underscore the requirement for strategies to mitigate potential adverse effects of romosozumab treatment on atherosclerosis and its related risk factors.

Red blood cell fatty acid patterns from 7 countries: Focus on the Omega-3 index.

Red blood cell (RBC) fatty acid (FA) patterns are becoming recognized as long-term biomarkers of tissue FA composition, but different analytical methods have complicated inter-study and international comparisons. Here we report RBC FA data, with a focus on the Omega-3 Index (EPA + DHA in% of total FAs in RBC), from samples of seven countries (USA, Canada, Italy, Spain, Germany, South Korea, and Japan) including 167,347 individuals (93% of all samples were from the US). FA data were generated by a uniform methodology from a variety of interventional and observational studies and from clinical laboratories. The cohorts differed in size, demographics, health status, and year of collection. Only the Canadian cohort was a formal, representative population-based survey. The mean Omega-3 Index of each country was categorized as desirable (>8%), moderate (>6% to 8%), low (>4% to 6%), or very low (≤4%). Only cohorts from Alaska (treated separately from the US), South Korea and Japan showed a desirable Omega-3 Index. The Spanish cohort had a moderate Omega-3 Index, while cohorts from the US, Canada, Italy, and Germany were all classified as low. This study is limited by the use of cohorts of convenience and small sample sizes in some countries. Countries undertaking national health status studies should utilize a uniform method to measure Omega-3 FA levels.

Circulating Sclerostin Levels Are Positively Related to Coronary Artery Disease Severity and Related Risk Factors.

Romosozumab is a newly available treatment for osteoporosis acting by sclerostin inhibition. Its cardiovascular safety has been questioned after finding excess cardiovascular disease (CVD)-related events in a pivotal phase 3 trial. Previous studies of relationships between circulating sclerostin levels and CVD and associated risk factors have yielded conflicting findings, likely reflecting small numbers and selected patient groups. We aimed to characterize relationships between sclerostin and CVD and related risk factors in more detail by examining these in two large cohorts, Ludwigshafen Risk and Cardiovascular Health study (LURIC; 34% female, mean age 63.0 years) and Avon Longitudinal Study of Parents and Children study (ALSPAC) mothers (mean age 48.1 years). Together these provided 5069 participants with complete data. Relationships between sclerostin and CVD risk factors were meta-analyzed, adjusted for age, sex (LURIC), body mass index, smoking, social deprivation, and ethnicity (ALSPAC). Higher sclerostin levels were associated with higher risk of diabetes mellitus (DM) (odds ratio [OR] = 1.25; 95% confidence interval [CI] 1.12, 1.37), risk of elevated fasting glucose (OR 1.15; CI 1.04, 1.26), and triglyceride levels (ß 0.03; CI 0.00, 0.06). Conversely, higher sclerostin was associated with lower estimated glomerular filtration rate (eGFR) (ß -0.20; CI -0.38, -0.02), HDL cholesterol (ß -0.05; CI -0.10, -0.01), and apolipoprotein A-I (ß -0.05; CI -0.08, -0.02) (difference in mean SD per SD increase in sclerostin, with 95% CI). In LURIC, higher sclerostin was associated with an increased risk of death from cardiac disease during follow-up (hazard ratio [HR] = 1.13; 1.03, 1.23) and with severity of coronary artery disease on angiogram as reflected by Friesinger score (0.05; 0.01, 0.09). Associations with cardiac mortality and coronary artery severity were partially attenuated after adjustment for risk factors potentially related to sclerostin, namely LDL and HDL cholesterol, log triglycerides, DM, hypertension, eGFR, and apolipoprotein A-I. Contrary to trial evidence suggesting sclerostin inhibition leads to an increased risk of CVD, sclerostin levels appear to be positively associated with coronary artery disease severity and mortality, partly explained by a relationship between higher sclerostin levels and major CVD risk factors. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Circulating uromodulin inhibits vascular calcification by interfering with pro-inflammatory cytokine signalling.

AIMS: Uromodulin is produced exclusively in the kidney and secreted into both urine and blood. Serum levels of uromodulin are correlated with kidney function and reduced in chronic kidney disease (CKD) patients, but physiological functions of serum uromodulin are still elusive. This study investigated the role of uromodulin in medial vascular calcification, a key factor associated with cardiovascular events and mortality in CKD patients. METHODS AND RESULTS: Experiments were performed in primary human (HAoSMCs) and mouse (MOVAS) aortic smooth muscle cells, cholecalciferol overload and subtotal nephrectomy mouse models and serum from CKD patients. In three independent cohorts of CKD patients, serum uromodulin concentrations were inversely correlated with serum calcification propensity. Uromodulin supplementation reduced phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. In human serum, pro-inflammatory cytokines tumour necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) co-immunoprecipitated with uromodulin. Uromodulin inhibited TNFα and IL-1ß-induced osteo-/chondrogenic signalling and activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated ß cells (NF-kB) as well as phosphate-induced NF-kB-dependent transcriptional activity in HAoSMCs. In vivo, adeno-associated virus (AAV)-mediated overexpression of uromodulin ameliorated vascular calcification in mice with cholecalciferol overload. Conversely, cholecalciferol overload-induced vascular calcification was aggravated in uromodulin-deficient mice. In contrast, uromodulin overexpression failed to reduce vascular calcification during renal failure in mice. Carbamylated uromodulin was detected in serum of CKD patients and uromodulin carbamylation inhibited its anti-calcific properties in vitro. CONCLUSIONS: Uromodulin counteracts vascular osteo-/chondrogenic transdifferentiation and calcification, at least in part, through interference with cytokine-dependent pro-calcific signalling. In CKD, reduction and carbamylation of uromodulin may contribute to vascular pathology.

Investigation of gene-gene interactions in cardiac traits and serum fatty acid levels in the LURIC Health Study.

Epistasis analysis elucidates the effects of gene-gene interactions (G×G) between multiple loci for complex traits. However, the large computational demands and the high multiple testing burden impede their discoveries. Here, we illustrate the utilization of two methods, main effect filtering based on individual GWAS results and biological knowledge-based modeling through Biofilter software, to reduce the number of interactions tested among single nucleotide polymorphisms (SNPs) for 15 cardiac-related traits and 14 fatty acids. We performed interaction analyses using the two filtering methods, adjusting for age, sex, body mass index (BMI), waist-hip ratio, and the first three principal components from genetic data, among 2,824 samples from the Ludwigshafen Risk and Cardiovascular (LURIC) Health Study. Using Biofilter, one interaction nearly met Bonferroni significance: an interaction between rs7735781 in XRCC4 and rs10804247 in XRCC5 was identified for venous thrombosis with a Bonferroni-adjusted likelihood ratio test (LRT) p: 0.0627. A total of 57 interactions were identified from main effect filtering for the cardiac traits G×G (10) and fatty acids G×G (47) at Bonferroni-adjusted LRT p < 0.05. For cardiac traits, the top interaction involved SNPs rs1383819 in SNTG1 and rs1493939 (138kb from 5' of SAMD12) with Bonferroni-adjusted LRT p: 0.0228 which was significantly associated with history of arterial hypertension. For fatty acids, the top interaction between rs4839193 in KCND3 and rs10829717 in LOC107984002 with Bonferroni-adjusted LRT p: 2.28×10-5 was associated with 9-trans 12-trans octadecanoic acid, an omega-6 trans fatty acid. The model inflation factor for the interactions under different filtering methods was evaluated from the standard median and the linear regression approach. Here, we applied filtering approaches to identify numerous genetic interactions related to cardiac-related outcomes as potential targets for therapy. The approaches described offer ways to detect epistasis in the complex traits and to improve precision medicine capability.

Intronic Variants in OCT1 are Associated with All-Cause and Cardiovascular Mortality in Metformin Users with Type 2 Diabetes.

PURPOSE: Organic cation transporters (Octs) use cations like endogenous compounds, toxins, and drugs, such as metformin, as substrates. Therefore, these proteins determine the pharmacokinetics and -dynamics of metformin and thus its efficacy. Of note, metformin is today the most commonly used pharmaceutical in the treatment of type 2 diabetes (T2DM) with nevertheless a great variability in clinical response, which attributes to genetic variances. The aim of this study was to determine the influence of intronic OCT1 SNPs on prevalence of all-cause and cardiovascular death. PATIENTS AND METHODS: Genotypes of 27 intronic SNPs in OCT1 were investigated in the LURIC study, a prospective cohort of 3316 participants scheduled for coronary angiography. We investigated whether these variants were associated with all-cause and cardiovascular death in 73 individuals with T2DM under metformin therapy, in individuals without diabetes, individuals with T2DM and individuals with T2DM without metformin therapy. RESULTS: In a multivariate Cox regression analysis adjusted for classical cardiovascular risk factors, 4 intronic OCT1 SNPs were significantly associated with all-cause and cardiovascular mortality in individuals with T2DM on metformin therapy. CONCLUSION: According to their OCT1 genotype, some individuals with T2DM on metformin therapy might be prone to an increased risk of cardiovascular death.

Phenome-wide association studies on cardiovascular health and fatty acids considering phenotype quality control practices for epidemiological data.

Phenome-wide association studies (PheWAS) allow agnostic investigation of common genetic variants in relation to a variety of phenotypes but preserving the power of PheWAS requires careful phenotypic quality control (QC) procedures. While QC of genetic data is well-defined, no established QC practices exist for multi-phenotypic data. Manually imposing sample size restrictions, identifying variable types/distributions, and locating problems such as missing data or outliers is arduous in large, multivariate datasets. In this paper, we perform two PheWAS on epidemiological data and, utilizing the novel software CLARITE (CLeaning to Analysis: Reproducibility-based Interface for Traits and Exposures), showcase a transparent and replicable phenome QC pipeline which we believe is a necessity for the field. Using data from the Ludwigshafen Risk and Cardiovascular (LURIC) Health Study we ran two PheWAS, one on cardiac-related diseases and the other on polyunsaturated fatty acids levels. These phenotypes underwent a stringent quality control screen and were regressed on a genome-wide sample of single nucleotide polymorphisms (SNPs). Seven SNPs were significant in association with dihomo-γ-linolenic acid, of which five were within fatty acid desaturases FADS1 and FADS2. PheWAS is a useful tool to elucidate the genetic architecture of complex disease phenotypes within a single experimental framework. However, to reduce computational and multiple-comparisons burden, careful assessment of phenotype quality and removal of low-quality data is prudent. Herein we perform two PheWAS while applying a detailed phenotype QC process, for which we provide a replicable pipeline that is modifiable for application to other large datasets with heterogenous phenotypes. As investigation of complex traits continues beyond traditional genome wide association studies (GWAS), such QC considerations and tools such as CLARITE are crucial to the in the analysis of non-genetic big data such as clinical measurements, lifestyle habits, and polygenic traits.

Mendelian Randomization Analysis Reveals a Causal Influence of Circulating Sclerostin Levels on Bone Mineral Density and Fractures.

In bone, sclerostin is mainly osteocyte-derived and plays an important local role in adaptive responses to mechanical loading. Whether circulating levels of sclerostin also play a functional role is currently unclear, which we aimed to examine by two-sample Mendelian randomization (MR). A genetic instrument for circulating sclerostin, derived from a genomewide association study (GWAS) meta-analysis of serum sclerostin in 10,584 European-descent individuals, was examined in relation to femoral neck bone mineral density (BMD; n = 32,744) in GEFOS and estimated bone mineral density (eBMD) by heel ultrasound (n = 426,824) and fracture risk (n = 426,795) in UK Biobank. Our GWAS identified two novel serum sclerostin loci, B4GALNT3 (standard deviation [SD]) change in sclerostin per A allele (ß = 0.20, p = 4.6 × 10-49 ) and GALNT1 (ß = 0.11 per G allele, p = 4.4 × 10-11 ). B4GALNT3 is an N-acetyl-galactosaminyltransferase, adding a terminal LacdiNAc disaccharide to target glycocoproteins, found to be predominantly expressed in kidney, whereas GALNT1 is an enzyme causing mucin-type O-linked glycosylation. Using these two single-nucleotide polymorphisms (SNPs) as genetic instruments, MR revealed an inverse causal relationship between serum sclerostin and femoral neck BMD (ß = -0.12, 95% confidence interval [CI] -0.20 to -0.05) and eBMD (ß = -0.12, 95% CI -0.14 to -0.10), and a positive relationship with fracture risk (ß = 0.11, 95% CI 0.01 to 0.21). Colocalization analysis demonstrated common genetic signals within the B4GALNT3 locus for higher sclerostin, lower eBMD, and greater B4GALNT3 expression in arterial tissue (probability >99%). Our findings suggest that higher sclerostin levels are causally related to lower BMD and greater fracture risk. Hence, strategies for reducing circulating sclerostin, for example by targeting glycosylation enzymes as suggested by our GWAS results, may prove valuable in treating osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.

Adjusted Troponin I for Improved Evaluation of Patients with Chest Pain.

The use of cardiac troponins (cTn) is the gold standard for diagnosing myocardial infarction. Independent of myocardial infarction (MI), however, sex, age and kidney function affect cTn levels. Here we developed a method to adjust cTnI levels for age, sex, and renal function, maintaining a unified cut-off value such as the 99th percentile. A total of 4587 individuals enrolled in a prospective longitudinal study were used to develop a model for adjustment of cTn. cTnI levels correlated with age and estimated glomerular filtration rate (eGFR) in males/females with rage = 0.436/0.518 and with reGFR = -0.142/-0.207. For adjustment, these variables served as covariates in a linear regression model with cTnI as dependent variable. This adjustment model was then applied to a real-world cohort of 1789 patients with suspected acute MI (AMI) (N = 407). Adjusting cTnI showed no relevant loss of diagnostic information, as evidenced by comparable areas under the receiver operator characteristic curves, to identify AMI in males and females for adjusted and unadjusted cTnI. In specific patients groups such as in elderly females, adjusting cTnI improved specificity for AMI compared with unadjusted cTnI. Specificity was also improved in patients with renal dysfunction by using the adjusted cTnI values. Thus, the adjustments improved the diagnostic ability of cTnI to identify AMI in elderly patients and in patients with renal dysfunction. Interpretation of cTnI values in complex emergency cases is facilitated by our method, which maintains a single diagnostic cut-off value in all patients.

Homoarginine-A prognostic indicator in adolescents and adults with complex congenital heart disease?

BACKGROUND: Homoarginine (hArg) has been shown to be of prognostic value in patients with chronic left heart failure. The present study aims to assess the clinical utility and prognostic value of hArg levels in patients with complex congenital heart disease (CHD). METHODS: Plasma hArg levels were measured in 143 patients with complex CHD and compared to clinical status, echocardiographic and laboratory parameters as well as the occurrence of adverse cardiac events. RESULTS: Median hArg levels were 1.5 µmol/l in CHD patients as compared to 1.70 µmol/l in healthy controls (p = 0.051). Median hArg levels were lowest in patients with Fontan palliation (1.27 µmol/l) and Eisenmenger physiology (0.99 µmol/l) and decreased with the severity of adverse cardiac events with lowest values found in patients prior to death or overt heart failure (0.89 µmol/l). According to ROC analysis, the most important predictors of adverse cardiac events were hArg levels (AUC 0.837, p<0.001, CI 0.726-0.947), NYHA class (AUC 0.800, p<0.001, CI 0.672-0.928) and NT-proBNP levels (AUC 0.780, p<0.001, CI 0.669-0.891). The occurrence of overt heart failure or death due to progressive heart failure were best predicted by NYHA class (AUC 0.945, p<0.001, CI 0.898-0.992), hArg levels (AUC 0.911, p<0.001, CI 0.850-0.971) and NT-proBNP levels (AUC 0.877, p<0.001, CI 0.791-0.962), respectively. CONCLUSION: In patients with complex CHD, hArg levels can predict adverse cardiac events as reliably as or even better than NT-proBNP levels and thus might be of prognostic value in this subset of patients.

Improved risk stratification in prevention by use of a panel of selected circulating microRNAs.

Risk stratification is crucial in prevention. Circulating microRNAs have been proposed as biomarkers in cardiovascular disease. Here a miR panel consisting of miRs related to different cardiovascular pathophysiologies, was evaluated to predict outcome in the context of prevention. MiR-34a, miR-223, miR-378, miR-499 and miR-133 were determined from peripheral blood by qPCR and combined to a risk panel. As derivation cohort, 178 individuals of the DETECT study, and as validation cohort, 129 individuals of the SHIP study were used in a case-control approach. Overall mortality and cardiovascular events were outcome measures. The Framingham Risk Score(FRS) and the SCORE system were applied as risk classification systems. The identified miR panel was significantly associated with mortality given by a hazard ratio(HR) of 3.0 (95% (CI): 1.09-8.43; p = 0.034) and of 2.9 (95% CI: 1.32-6.33; p = 0.008) after adjusting for the FRS in the derivation cohort. In a validation cohort the miR-panel had a HR of 1.31 (95% CI: 1.03-1.66; p = 0.03) and of 1.29 (95% CI: 1.02-1.64; p = 0.03) in a FRS/SCORE adjusted-model. A FRS/SCORE risk model was significantly improved to predict mortality by the miR panel with continuous net reclassification index of 0.42/0.49 (p = 0.014/0.005). The present miR panel of 5 circulating miRs is able to improve risk stratification in prevention with respect to mortality beyond the FRS or SCORE.

The Proline 7 Substitution in the Preproneuropeptide Y Is Associated with Higher Hepatic Lipase Activity In Vivo.

Hepatic lipase (HL) functions as a lipolytic enzyme that hydrolyzes triglycerides and phospholipids present in circulating plasma lipoproteins. Plasma HL activity is known to be regulated by hormonal and metabolic factors, but HL responsiveness to insulin as well as its role in modulating atherosclerotic risk is still controversial. We investigated on the influence of a known polymorphism in the neurotransmitter neuropeptide Y (NPY) on HL activity in two different cohorts consisting of diabetic and nondiabetic patients. HL activity was 24% and 34% higher on nondiabetic and diabetic subjects in the presence of the 7Pro allele in NPY, respectively. The presence of the 7Pro allele was an independent predictor of HL activity in multivariate analyses in both cohorts. These data suggest a regulatory effect of NPY on HL activity. Among carriers of the 7Pro allele, we also found a statistically significant lower absolute number of infarctions compared to noncarriers (p < 0.05) and a nonsignificant trend towards less myocardial infarction in the 7Pro allele diabetic carriers (p = 0.085). In conclusion, the common 7Pro allele in NPY was associated with higher HL activity in nondiabetic and diabetic subjects and its presence seems to coincide with a lower frequency of certain cardiovascular events.

Galectin-3 binding protein, coronary artery disease and cardiovascular mortality: Insights from the LURIC study.

BACKGROUND AND AIMS: Galectin-3 binding protein (Gal-3BP) has been associated with inflammation and cancer, however, its role in coronary artery disease (CAD) and cardiovascular outcome remains unclear. METHODS: Gal-3BP plasma levels were measured by ELISA in 2922 individuals from the LURIC study (62.7 ± 10.6 years, 62.7% male). All-cause and cardiovascular mortality was assessed by Kaplan-Meier analysis and Cox proportional hazards regression. Causal involvement of Gal-3BP was tested for by Mendelian randomization. Gal-3BP effects on human monocyte-derived macrophages were assessed in vitro. RESULTS: During 8.8 ± 3.0 years, 866 individuals died, 654 of cardiovascular causes. There was a significant increase in all-cause and cardiovascular mortality with increasing Gal-3BP quintiles. After thorough adjustment, all-cause mortality remained significantly increased in the fifth Gal-3BP quintile (HRQ5 1.292 (1.030-1.620), p = 0.027); cardiovascular mortality remained increased in Gal-3BP quintiles two to five (HRQ51.433 (1.061-1.935, p = 0.019). Gal-3BP levels were not associated with diagnosis and extent of coronary artery disease. In addition, Mendelian randomization did not show a direct causal relationship between Gal-3BP levels and mortality. Gal-3BP levels were, however, independently associated with markers of metabolic and inflammatory distress. In vitro, Gal-3BP induced a pro-inflammatory response in human monocyte-derived macrophages. Adding Gal-3BP levels to the ESC score improved risk assessment in patients with ESC SCORE-based risk >5% (p = 0.010). CONCLUSIONS: In a large clinical cohort of CAD patients, Gal-3BP levels are independently associated with all-cause and cardiovascular mortality. The underlying mechanisms may likely involve metabolic and inflammatory distress. To further evaluate the potential clinical value of Gal-3BP, prospective studies are needed.

Lipoprotein Apheresis for Lipoprotein(a)-Associated Cardiovascular Disease: Prospective 5 Years of Follow-Up and Apolipoprotein(a) Characterization.

OBJECTIVE: Lipoprotein(a)-hyperlipoproteinemia (Lp(a)-HLP) along with progressive cardiovascular disease has been approved as indication for regular lipoprotein apheresis (LA) in Germany since 2008. We aimed to study the long-term preventive effect of LA and to assess hypothetical clinical correlations of apolipoprotein(a) (apo(a)) by analyzing genotypes and phenotypes. APPROACH AND RESULTS: This prospective observational multicenter study included 170 patients with Lp(a)-HLP and progressive cardiovascular disease (48.9 years median age at diagnosis) despite other cardiovascular risk factors, including low-density lipoprotein cholesterol had maximally been treated (mean baseline low-density lipoprotein cholesterol: measured, 2.56 mmol/L [98.9 mg/dL] and corrected, 1.72 mmol/L [66.3 mg/dL]). Patients were prospectively investigated during a 5-year period about annual incidence rates of cardiovascular events. In addition, apo(a) isoforms and polymorphisms at the apo(a) gene (LPA) were characterized. One hundred fifty-four patients (90.6%) completed 5 years of follow-up. Mean Lp(a) concentration before commencing regular LA was 108.1 mg/dL. This was reduced by a single LA treatment by 68.1% on average. Significant decline of the mean annual cardiovascular event rate was observed from 0.58±0.53 2 years before regular LA to 0.11±0.15 thereafter (P<0.0001); 95.3% of patients expressed at least 1 small apo(a) isoform. Small apo(a) isoform (35.2%) carrying phenotypes were not tagged by single-nucleotide polymorphisms rs10455872 or rs3798220. CONCLUSIONS: Results of 5 years of prospective follow-up confirm that LA has a lasting effect on prevention of cardiovascular events in patients with Lp(a)-HLP. Patients clinically selected by progressive cardiovascular disease were characterized by a highly frequent expression of small apo(a) isoforms. Only Lp(a) concentration seemed to comprehensively reflect Lp(a)-associated cardiovascular risk, however.

Augmentation of phosphate-induced osteo-/chondrogenic transformation of vascular smooth muscle cells by homoarginine.

AIMS: Reduced homoarginine plasma levels are associated with unfavourable cardiovascular outcome in chronic kidney disease (CKD). Cardiovascular events in CKD are fostered by vascular calcification, an active process promoted by hyperphosphatemia and involving osteo-/chondrogenic transformation of vascular smooth muscle cells (VSMCs). The present study explored the effect of homoarginine on phosphate-induced osteo-/chondrogenic signalling and vascular calcification. METHODS AND RESULTS: Experiments were performed in hyperphosphatemic klotho-hypomorphic mice (kl/kl), in subtotal nephrectomy and vitamin D3-overload mouse calcification models and in primary human aortic smooth muscle cells (HAoSMCs). As a result, plasma homoarginine levels were lower in kl/kl mice than in wild-type mice and in both genotypes significantly increased by lifelong treatment with homoarginine. Surprisingly, homoarginine treatment of kl/kl mice and of mice with renal failure after subtotal nephrectomy augmented vascular calcification and enhanced the transcript levels of plasminogen activator inhibitor 1 (Pai1) and of osteogenic markers Msx2, Cbfa1, and Alpl. Similarly, homoarginine treatment of HAoSMCs increased phosphate-induced calcium deposition, ALP activity, as well as PAI1, MSX2, CBFA1, and ALPL mRNA levels. Homoarginine alone up-regulated osteo-/chondrogenic signalling and indicators of oxidative stress in HAoSMCs. Furthermore, homoarginine reduced citrulline formation from arginine by nitric oxide (NO) synthase (NOS) isoforms. NO formation by NOS was reduced when using homoarginine as a substrate instead of arginine. The osteoinductive effects of homoarginine were mimicked by NOS inhibitor L-NAME and abolished by additional treatment with the NO donors DETA-NONOate and PAPA-NONOate or the antioxidants TEMPOL and TIRON. Furthermore, homoarginine augmented vascular calcification and aortic osteo-/chondrogenic signalling in mice after vitamin D3-overload, effects reversed by the NO donor molsidomine. CONCLUSION: Homoarginine augments osteo-/chondrogenic transformation of VSMCs and vascular calcification, effects involving impaired NO formation from homoarginine.

Inverse association of the endogenous thrombin potential (ETP) with cardiovascular death: the Ludwigshafen Risk and Cardiovascular Health (LURIC) study.

BACKGROUND: Coagulation and prothrombotic potential have genuinely been associated with increased cardiovascular risk. However, not all studies in this regard are conclusive. Some clinical trials have shown an increased frequency of cardiovascular complications in patients receiving direct thrombin inhibitors. Previous data from human subjects after acute cardiovascular events showed an inverse association between the thrombin generation marker F1+2 and cardiovascular endpoints indicating that not the lowest, but a slightly elevated propensity for thrombin generation is associated with a lower risk of cardiovascular events. This observation has been supported by findings in animal models of atherosclerosis. Hence, we evaluated the association between the endogenous thrombin potential (ETP) and cardiovascular death (CVD) and markers of vascular dysfunction in a large prospective study with long-term follow up. METHOD: After excluding patients receiving anticoagulants we tested ETP in 2196 participants (median follow-up 10 years) for its ability to predict vascular death (CVD). In addition, the association between ETP and sVCAM-1, sICAM-1, LpPLA2, hsCRP and SAA was determined. RESULTS: We observed an inverse association between ETP and CVD with the lowest hazard ratio in the 4th ETP quartile. The nadirs of sICAM-1 or sVCAM-1 were observed in the 3rd, for LpPLA2 in the 4th ETP quartile. Conversely, hsCRP and SAA were highest in the 4th quartile. CONCLUSIONS: These results demonstrate that not the lowest ETP possible, but slightly higher levels are associated with a reduced risk of CVD and lower markers of endothelial dysfunction, suggesting a more complex role of thrombin in cardiovascular disease.

Increased soluble ST2 predicts long-term mortality in patients with stable coronary artery disease: results from the Ludwigshafen risk and cardiovascular health study.

BACKGROUND: Soluble suppression of tumorigenicity 2 (sST2) has emerged as a strong prognostic biomarker in patients with heart failure and myocardial infarction. The aim of this study was to evaluate the long-term prognostic value of sST2 in patients with stable coronary artery disease (CAD). METHODS: sST2 plasma concentrations were measured in 1345 patients with stable CAD referred for coronary angiography at a single tertiary care center. The primary endpoint was all-cause mortality. RESULTS: During a median follow-up time of 9.8 years, 477 (36%) patients died. The median sST2 plasma concentration at baseline was significantly higher among decedents than survivors (21.4 vs 18.5 ng/mL; P < 0.001). In multivariate Cox proportional hazards regression analysis, sST2 was an independent predictor of all-cause mortality (risk ratio 1.16 per 1-SD increase in log-transformed values; 95% CI 1.05-1.29; P = 0.004). In the same multivariate analysis, amino-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hs-cTnT) were also independent predictors, whereas galectin-3 was not. Patients with sST2 in the highest quartile (>24.6 ng/mL) displayed a 2-fold increased risk of death in univariate analysis, which was attenuated but remained significant in a fully adjusted model (risk ratio 1.39; 95% CI 1.10-1.76; P = 0.006). Further analysis showed that the prognostic impact of sST2 was additive to NT-proBNP and hs-cTnT. Using a multibiomarker approach combining these 3 complementary makers, we demonstrated that patients with all 3 biomarkers in the highest quartiles had the poorest outcome. CONCLUSIONS: In this cohort of patients with stable CAD, increased sST2 was an independent predictor of long-term all-cause mortality and provided complementary prognostic information to hs-cTnT and NT-proBNP.

Glycerophospholipid and sphingolipid species and mortality: the Ludwigshafen Risk and Cardiovascular Health (LURIC) study.

Vascular and metabolic diseases cause half of total mortality in Europe. New prognostic markers would provide a valuable tool to improve outcome. First evidence supports the usefulness of plasma lipid species as easily accessible markers for certain diseases. Here we analyzed association of plasma lipid species with mortality in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. Plasma lipid species were quantified by electrospray ionization tandem mass spectrometry and Cox proportional hazards regression was applied to assess their association with total and cardiovascular mortality. Overall no differences were detected between total and cardiovascular mortality. Highly polyunsaturated phosphatidylcholine species together with lysophosphatidylcholine species and long chain saturated sphingomyelin and ceramide species seem to be associated with a protective effect. The predominantly circulating phosphatidylcholine-based as well as phosphatidylethanolamine-based ether species and phosphatidylethanolamine species were positively associated with total and cardiovascular mortality. Saturated and monounsaturated phosphatidylcholine species, especially phosphatidylcholine 32∶0 (most probably dipalmitoyl-phosphatidylcholine) and palmitate containing sphingomyelin and ceramide species showed together with 24∶1 containing sphingomyelin and ceramide species strongest positive association with mortality. A quotient of the sums of the six most protective species and the six species with the strongest positive mortality association indicated an almost 3-fold increased risk of mortality, which was higher than the hazard ratio for known risk factors in our cohort. Plasma lipid species levels and especially ratios of certain species may be valuable prognostic marker for cardiovascular and total mortality.

Plant sterols and plant stanols in the management of dyslipidaemia and prevention of cardiovascular disease.

OBJECTIVE: This EAS Consensus Panel critically appraised evidence relevant to the benefit to risk relationship of functional foods with added plant sterols and/or plant stanols, as components of a healthy lifestyle, to reduce plasma low-density lipoprotein-cholesterol (LDL-C) levels, and thereby lower cardiovascular risk. METHODS AND RESULTS: Plant sterols/stanols (when taken at 2 g/day) cause significant inhibition of cholesterol absorption and lower LDL-C levels by between 8 and 10%. The relative proportions of cholesterol versus sterol/stanol levels are similar in both plasma and tissue, with levels of sterols/stanols being 500-/10,000-fold lower than those of cholesterol, suggesting they are handled similarly to cholesterol in most cells. Despite possible atherogenicity of marked elevations in circulating levels of plant sterols/stanols, protective effects have been observed in some animal models of atherosclerosis. Higher plasma levels of plant sterols/stanols associated with intakes of 2 g/day in man have not been linked to adverse effects on health in long-term human studies. Importantly, at this dose, plant sterol/stanol-mediated LDL-C lowering is additive to that of statins in dyslipidaemic subjects, equivalent to doubling the dose of statin. The reported 6-9% lowering of plasma triglyceride by 2 g/day in hypertriglyceridaemic patients warrants further evaluation. CONCLUSION: Based on LDL-C lowering and the absence of adverse signals, this EAS Consensus Panel concludes that functional foods with plant sterols/stanols may be considered 1) in individuals with high cholesterol levels at intermediate or low global cardiovascular risk who do not qualify for pharmacotherapy, 2) as an adjunct to pharmacologic therapy in high and very high risk patients who fail to achieve LDL-C targets on statins or are statin- intolerant, 3) and in adults and children (>6 years) with familial hypercholesterolaemia, in line with current guidance. However, it must be acknowledged that there are no randomised, controlled clinical trial data with hard end-points to establish clinical benefit from the use of plant sterols or plant stanols.

Association between a gene variant near ataxia telangiectasia mutated and coronary artery disease in men.

OBJECTIVE: Type 2 diabetes is accompanied by increased mortality from coronary artery disease (CAD), but the mechanisms linking these conditions remain elusive. Hence, treatment of hyperglycaemia alone is not sufficient to avoid CAD in diabetes. Alternative views suggest that metabolic and vascular diseases share unifying cellular defects that could serve as targets for novel therapeutic strategies. Recently, a variant [single-nucleotide polymorphism (SNP); rs11212617] near the gene for ataxia telangiectasia mutated (ATM) has been associated with glycaemic response to metformin. MATERIALS AND METHODS: We determined rs11212617 in 240 male patients who underwent elective coronary angiography. RESULTS: While the variant was not associated with glucose concentrations, the A allele was significantly associated with the presence of CAD (chi-square, p = 0.003), as well as with logarithmically transformed quantitative CAD indices [severe score (SS): 0.5 (0.4-0.6) vs 0.3 (0.2-0.5); extent score (ES): 2.63 (2.4-2.9) vs 1.94 (1.4-2.4), both p < 0.05, respectively]. Multivariate analysis revealed an independent association between the A allele with ES (ß = 0.17, p < 0.01). CONCLUSION: Our data suggest that ATM-dependent signalling might play a role in the development of atherosclerotic vascular disease, but larger studies are necessary to substantiate such a hypothesis.