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Circulating cell-free microRNAs (miRNAs) represent a major reservoir for biomarker discovery. Unfortunately, their implementation in clinical practice is limited due to a profound lack of reproducibility. The great technical variability linked to major pre-analytical and analytical caveats makes the interpretation of circulating cell-free miRNA data challenging and leads to inconsistent findings. Additional efforts directed to standardization are fundamental. Several well-established protocols are currently used by independent groups worldwide. Nonetheless, there are some specific aspects in specimen collection and processing, sample handling, miRNA quantification, and data analysis that should be considered to ensure reproducibility of results. Here, we have addressed this challenge using an alternative approach. We have highlighted and discussed common pitfalls that negatively impact the robustness of circulating miRNA quantification and their application for clinical decision-making. Furthermore, we provide a checklist usable by investigators to facilitate and ensure the control of the whole miRNA quantification and analytical process. We expect that these recommendations improve the reproducibility of findings, and ultimately, facilitate the incorporation of circulating miRNA profiles into clinical practice as the next generation of disease biomarkers.

Supplemental Digital Content is available in the text., RAASi (renin-angiotensin-aldosterone system inhibitors) are suggested as possible treatment option in the early phase of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. A meta-analysis investigating the possible detrimental effects of RAASi on the severity of (SARS-CoV-2) infection showed that ambulatory use of RAASi, by hospitalized patients, has a neutral effect. It is, however, conceivable that this observation is biased by the fact that antihypertensive medications, are often discontinued at or during admission in hospitalized patients with SARS-CoV-2. We, therefore, investigated the effect of discontinuation of antihypertensive medications, in hospitalized patients with SARS-CoV-2. We performed a retrospective observational study on 1584 hospitalized patients with SARS-CoV-2 from 10 participating hospitals in the Netherlands. Differences in the outcome (severity of disease or death) between the groups in which medications were either continued or discontinued during the course of hospitalization were assessed using logistic regression models. Discontinuation of angiotensin receptor blockers, ACE (angiotensin-converting enzyme) inhibitors and β-blockers, even when corrected for sex, age, and severity of symptoms during admission, resulted in a 2 to 4× higher risk of dying from SARS-CoV-2 infection (odds ratio [95% CI]); angiotensin receptor blockers 2.65 [1.17–6.04], ACE inhibitor (2.28 [1.15–4.54]), and β-blocker (3.60 [1.10–10.27]). In conclusion, discontinuation of at-home ACE inhibitor, angiotensin receptor blockers, or β-blocker in patients hospitalized for a SARS-CoV-2 infection was associated with an increased risk of dying, whereas discontinuation of calcium channel blockers and diuretics was not.

Currently, risk prediction models like SCORE are used for decision making in the primary prevention of cardiovascular disease. The external validity of these models is questionable since they give rise to overtreatment with statins or antihypertensive drugs. Detailed individual risk assessment may reduce this drawback and will increase cost effectiveness. The CT derived coronary calcium score, in asymptomatic patients, was shown to be more accurate than the current prediction models. A coronary calcium score of zero reclassifies a significant number of individuals to a lower risk group and subsequently prevent overtreatment. Using this strategy, it can be anticipated that the Dutch healthcare costs can be reduced by at least 15 million Euro per year.

The role of antihypertensives, especially Renin–Angiotensin–Aldosterone System inhibitors, is still debatable in COVID‐19‐related severity and outcome. Therefore, we search for a more global analysis of antihypertensive medication in relation to SAS‐CoV‐2 severity using prescription data worldwide. The association between the percentage use of different types of antihypertensive medications and mortality rates due to a SARS‐CoV‐2 infection during the first 3 weeks of the pandemic was analyzed using random effects linear regression models for 30 countries worldwide. Higher percentages of prescribed angiotensin receptor blockers (ARBs) (β, 95% confidence interval [CI]; −0.02 [−0.04 to −0.0012]; p = .042) and calcium channel blockers (CCBs) (β, 95% CI; −0.023 [−0.05 to −0.0028]; p = .0304) were associated with a lower first 3‐week SARS‐CoV‐2‐related death rate, whereas a higher percentage of prescribed angiotensin‐converting enzyme inhibitors (ACEis) (β, 95% CI; 0.03 [0.0061–0.05]; p = .0103) was associated with a higher first 3‐week death rate, even when adjusted for age and metformin use. There was no association between the amount of prescribed beta‐blockers (BBs) and diuretics (Diu) and the first 3‐week death rate. When analyzing the combination of drugs that is used by at least 50% of antihypertensive users, within the different countries, countries with the lowest first 3‐week death rates had at least an angiotensin receptor blocker as one of the most often prescribed antihypertensive medications (ARBs/CCBs: [β, 95% CI; −0.02 [−0.03 to −0.004]; p = .009], ARBs/BBs: [β, 95% CI; −0.03 [−0.05 to −0.006]; p = .01]). Finally, countries prescribing high‐potency ARBs had lower first 3‐week ARBs. In conclusion, ARBs and CCB seem to have a protective effect against death from SARS‐CoV‐2 infection., Highlights Higher percentages of prescribed angiotensin receptor blockers and calcium channel blockers were associated with a lower first 3‐week SARS‐CoV‐2‐related death rate.Higher percentage of prescribed angiotensin‐converting enzyme was associated with a higher first 3‐week death rates.Countries with the lowest first 3‐week death rates had at least an angiotensin receptor blocker as one of the most often prescribed antihypertensive medications.

The SARS-CoV-2 pandemic is a healthcare crisis caused by insufficient knowledge applicable to effectively combat the virus. Therefore, different scientific discovery strategies need to be connected, to generate a rational treatment which can be made available as rapidly as possible. This relies on a solid theoretical understanding of the mechanisms of SARS-CoV-2 infection and host responses, which is coupled to the practical experience of clinicians that are treating patients. Because SARS-CoV-2 enters the cell by binding to angiotensin-converting enzyme 2 (ACE2), targeting ACE2 to prevent such binding seems an obvious strategy to combat infection. However, ACE2 performs its functions outside the cell and was found to enter the cell only by angiotensin II type 1 receptor (AT1R)-induced endocytosis, after which ACE2 is destroyed. This means that preventing uptake of ACE2 into the cell by blocking AT1R would be a more logical approach to limit entry of SARS-CoV-2 into the cell. Since ACE2 plays an important protective role in maintaining key biological processes, treatments should not disrupt the functional capacity of ACE2, to counterbalance the negative effects of the infection. Based on known mechanisms and knowledge of the characteristics of SARS-CoV we propose the hypothesis that the immune system facilitates SARS-CoV-2 replication which disrupts immune regulatory mechanisms. The proposed mechanism by which SARS-CoV-2 causes disease immediately suggests a possible treatment, since the AT1R is a key player in this whole process. AT1R antagonists appear to be the ideal candidate for the treatment of SARS-CoV-2 infection. AT1R antagonists counterbalance the negative consequences of angiotesnin II and, in addition, they might even be involved in preventing the cellular uptake of the virus without interfering with ACE2 function. AT1R antagonists are widely available, cheap, and safe. Therefore, we propose to consider using AT1R antagonists in the treatment of SARS-CoV-2.

Cardiometabolic disease (CMD), such as type 2 diabetes mellitus and cardiovascular disease, contributes significantly to morbidity and mortality on a global scale. The gut microbiota has emerged as a potential target to beneficially modulate CMD risk, possibly via dietary interventions. Dietary interventions have been shown to considerably alter gut microbiota composition and function. Moreover, several diet-derived microbial metabolites are able to modulate human metabolism and thereby alter CMD risk. Dietary interventions that affect gut microbiota composition and function are therefore a promising, novel, and cost-efficient method to reduce CMD risk. Studies suggest that fermentable carbohydrates can beneficially alter gut microbiota composition and function, whereas high animal protein and high fat intake negatively impact gut microbiota function and composition. This review focuses on the role of macronutrients (i.e., carbohydrate, protein, and fat) and dietary patterns (e.g., vegetarian/vegan and Mediterranean diet) in gut microbiota composition and function in the context of CMD.

Background Because of a nonresponse to aspirin (aspirin resistance), patients with acute coronary syndrome (ACS) are at increased risk of developing recurrent event. The in vitro platelet function tests have potential limitations, making them unsuitable for the detection of aspirin resistance. We investigated whether miR‐19b‐1‐5p could be utilized as a biomarker for aspirin resistance and future major adverse cardio‐cerebrovascular (MACCE) events in patients with ACS. Methods and Results In this cohort study, patients with ACS were enrolled from multiple tertiary hospitals in Christchurch, Hong Kong, Sarawak, and Singapore between 2011 and 2015. MiR‐19b‐1‐5p expression was measured from buffy coat of patients with ACS (n=945) by reverse transcription quantitative polymerase chain reaction. Platelet function was determined by Multiplate aggregometry testing. MACCE was collected over a mean follow‐up time of 1.01±0.43 years. Low miR‐19b‐1‐5p expression was found to be related to aspirin resistance as could be observed from sustained platelet aggregation in the presence of aspirin (‐Log‐miR‐19b‐1‐5p, [unstandardized beta, 44.50; 95% CI, 2.20–86.80; P P P =0.01) on the risk of MACCE. Conclusions Lower miR‐19b‐1‐5p expression was found to be associated with sustained platelet aggregation on aspirin, and a higher risk of MACCE in patients with ACS. Therefore, miR‐19b‐1‐5p could be a suitable marker for aspirin resistance and might predict recurrence of MACCE in patients with ACS.

Background Aortic valve calcium (AVC) is an important hallmark of aortic valve stenosis, which is the most common valvular heart disease in the Western world. Studies suggesting an important role for lipoprotein(a) [Lp(a)] in the etiology of AVC are accumulating, yet population-based evidence is scarce. Therefore, we investigated the association of Lp(a) with the presence of AVC in two large cohorts. Methods A total of 2412 participants from the population-based Rotterdam Study (52% women, mean age=69.6±6.3), and 859 asymptomatic persons from the Amsterdam Medical Center outpatient clinic for familial premature (non-valvular) atherosclerosis (57%women, mean age=45.9±11.6) underwent blood sampling to determine serum Lp(a) and non-enhanced cardiac CT-scan to assess AVC. We combined both cohorts and investigated the association of Lp(a) with the presence and amount of AVC using logistic and linear regression models, adjusting for age, sex, smoking, body mass index, non-high density lipoprotein cholesterol and use of antihypertensive medication. Results Out of a total of 3271 subjects with an average age of 63.4±7.98, AVC was present in 844 (25.8%) individuals. Higher levels of Lp(a) were associated with the presence of AVC, independent of age, sex and cardiovascular risk factors [Odds ratio (OR) per 1-SD increase in Lp(a): 1.39 (95% CI:1.27; 1.51). In persons with AVC, a higher level of Lp(a) was also related to larger volume of AVC [β per 1-SD increase in Lp(a): 0.76 (95% CI:1.27; 1.51)]. All findings were similar across both cohorts. Conclusion Lp(a) is a prominent and independent marker of the presence and amount of AVC in the general population. Future studies investigating the effect of Lp(a) lowering on the progression of AVC are warranted. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): NESTOR program for geriatric research, the Netherlands Heart Foundation, the Netherlands Organization for Scientific Research, the Health Research and Development Council (28-2975 and 97-1-364), and the Municipality of Rotterdam

Background There is much debate on the use of angiotensin receptor blockers (ARBs) in severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2)-infected patients. Although it has been suggested that ARBs might lead to a higher susceptibility and severity of SARS-CoV-2 infection, experimental data suggest that ARBs may reduce acute lung injury via blocking angiotensin-II-mediated pulmonary permeability, inflammation, and fibrosis. However, despite these hypotheses, specific studies on ARBs in SARS-CoV-2 patients are lacking.Methods The PRAETORIAN-COVID trial is a multicenter, double-blind, placebo-controlled 1:1 randomized clinical trial in adult hospitalized SARS-CoV-2-infected patients (n = 651). The primary aim is to investigate the effect of the ARB valsartan compared to placebo on the composite end point of admission to an intensive care unit, mechanical ventilation, or death within 14 days of randomization. The active-treatment arm will receive valsartan in a dosage titrated to blood pressure up to a maximum of 160 mg bid, and the placebo arm will receive matching placebo. Treatment duration will be 14 days, or until the occurrence of the primary end point or until hospital discharge, if either of these occurs within 14 days. The trial is registered at clinicaltrials.gov (NCT04335786, 2020).Summary The PRAETORIAN-COVID trial is a double-blind, placebo-controlled 1:1 randomized trial to assess the effect of valsartan compared to placebo on the occurrence of ICU admission, mechanical ventilation, and death in hospitalized SARS-CoV-2-infected patients. The results of this study might impact the treatment of SARS-CoV-2 patients globally.

Background Age and sex based coronary artery calcium score (CAC) percentiles have been used to improve coronary artery disease (CAD) risk prediction. However, the main limitation of the CACs percentiles currently in use is that they are often based on single studies. We performed a pooled analysis of all available studies that reported on CAC percentiles, in order to develop more generalizable age and sex nomograms. Methods PubMed/Medline and Embase were searched for studies that reported nomograms of age and sex-based CACs percentiles. Studies were included if they reported data collected among asymptomatic individuals without a history of cardiovascular disease. Absolute CACs for each specific percentile stratum were pooled and new percentiles were generated taking into account the sample size of the study. Results We found 831 studies, of which 12 met the inclusion criteria. Data on CACs percentiles of 134,336 Western and 33,488 Asians were pooled separately, rendering a weighted CACs percentile nomogram available at https://www.calciumscorecalculator.com . Our weighted percentiles differed by up to 24% from the nomograms in use today. Conclusions Our pooled age and sex based CACs percentiles based on over 155,000 individuals should provide a measure of risk that is more applicable to a wider population than the ones currently in use and hopefully will lead to better risk assessment and treatment decisions.

Objective: Netrin-1 has been shown to play a role in the initiation of atherosclerosis in mice models. However, little is known about the role of Netrin-1 in humans. We set out to study whether Netrin-1 is associated with different stages of atherosclerosis. Approach and Results: Plasma Netrin-1 levels were measured in different patient cohorts: (1) 22 patients with high cardiovascular risk who underwent arterial wall inflammation assessment using positron-emission tomography / computed tomography, (2) 168 patients with a positive family history of premature atherosclerosis in whom coronary artery calcium scores were obtained, and (3) 104 patients with chest pain who underwent coronary computed tomography angiography imaging to evaluate plaque vulnerability and burden. Netrin-1 plasma levels were negatively correlated with arterial wall inflammation (β, −0.01 [95% CI, 0.02 to −0.01] R 2 , 0.61; P P P =0.511). However, Netrin-1 plasma levels were negatively correlated to total plaque volume (β, −0.09 [95% CI, −0.11 to −0.08] R 2 , 0.57, P R 2 , 0.53; P R 2 , 0.41; P ConclusionS: Netrin-1 plasma levels are lower in patients with subclinical atherosclerosis and in patients with arterial wall inflammation. Netrin-1 is not associated with plaque vulnerability; however, it is negatively correlated to plaque burden, suggesting that Netrin-1 is involved in some, but not all, stages of atherosclerosis.

BACKGROUND In the past decade, the search for circulating microRNA (miRNA) biomarkers has yielded numerous associations between miRNAs and different types of disease. However, many of these relations could not be replicated in subsequent studies under similar experimental conditions. Although this lack of replicability may be explained by the variation in experimental design and analysis methods, guidelines on the most appropriate design and analysis methods to study circulating miRNAs are scarce. CONTENT miRNA biomarker experiments generally consist of a discovery phase and a validation phase. In the discovery phase, typically hundreds of miRNAs are measured in parallel to identify candidate biomarkers. Because of the costs of such high-throughput experiments, the number of individuals included in those studies is often too small, which can easily lead to false positives and false negatives. In the validation phase, a small number of identified biomarker candidates are measured in a large cohort of cases and controls, generally by quantitative PCR (qPCR). Although qPCR is a sensitive method to measure miRNAs in the circulation, experimental design and qPCR data analysis remain challenging. Omitting some crucial steps in the design and analysis of the qPCR experiment or performing them incorrectly can cause serious biases, ultimately leading to false conclusions. SUMMARY In this review, we aim to expose and discuss the most common sources of interstudy variation in miRNA research from a methodological point of view and to provide guidelines on how to perform these steps correctly to increase replicability of studies on circulating miRNAs.

Aims Small studies suggested circulating microRNAs (miRNAs) as biomarkers for heart failure (HF). However, standardized approaches and quality assessment for measuring circulating miRNAs are not uniformly established, and most studies have been small, so that results are inconsistent. We used a standardized data handling protocol, optimized for circulating miRNA qPCRs to remove noise and used it to assess which circulating miRNAs robustly add prognostic information in patients with HF. Methods and results We measured 12 miRNAs in two independent cohorts totalling 2203 subjects. Cohort I (Barcelona) comprised 834 chronic HF patients. Cohort II (Detroit) comprised 1369 chronic HF patients. Each sample was measured in duplicate, and normalized to a very abundant and stable miRNA (miR-486-5p). We used a multistep algorithm to distinguish false amplification signals and thus classify each miRNA measurement as ‘valid’, ‘undetectable’ or ‘invalid’. Higher levels of miR-1254 and miR-1306-5p were significantly associated with risk of the combined endpoint of all-cause mortality and HF hospitalization in both cohorts, with hazard ratios ranging from 1.11 to 1.21 per log increase (P-values 0.004 to 0.009). However, adding these miRNAs to established predictors (age, sex, haemoglobin, renal function, and NT-proBNP) did not further augment the c-statistic beyond 0.69 (cohort I) or 0.70 (cohort II). Conclusion We used a stringent quality assessment for miRNA testing, and were able to replicate the association of miR-1254 and miR-1306-5p with risk of death and HF hospitalization in HF patients of two independent cohorts. However, these two circulating miRNAs failed to improve prognostication over established predictors.

Background and aims: The risk of developing cardiovascular disease (CVD) is twice as high among smoking individuals compared to non-smokers. Monocytes are involved in smoking-related atherosclerotic plaque formation. In this study, we investigated whether smokers with an increased risk of developing CVD can be identified on the basis of monocyte-derived miRNA expression levels. Methods: We performed a miRNA microarray experiment on isolated monocytes from smoking, former smoking and non-smoking individuals in a cohort of patients with premature CVD and healthy controls (Cohort I, n = 76). Results: We found miR-124-3p to be heterogeneously expressed among all smoking individuals, whereas expression was low in non-smokers. Subsequently, RT-qPCR measurements on whole blood showed that among smoking individuals an increase in miR-124-3p is associated with an increased risk for advanced atherosclerotic disease (cohort II, n = 24) (OR 11.72 95% CI 1.09-126.53) and subclinical atherosclerosis (coronary artery calcium score >= 80th percentile, cohort III n = 138) (OR 2.71, 95% CI 1.05e7.01). This was not observed among former smokers or non-smoking individuals. Flow cytometric analysis demonstrated that high miR-124-3p expression was associated with upregulation of the monocyte surface markers CD45RA, CD29 and CD206, indicating an altered monocyte phenotype. Finally, overexpression of miR-124-3p resulted in an upregulation of CD206 surface expression on monocytes. Conclusions: High miR-124-3p expression is associated with an increased risk of subclinical atherosclerosis in smoking individuals and with an altered monocyte phenotype. This may suggest that miR-124-3p identifies which smoking individuals are susceptible to the atherogenic effects of smoking. (C) 2017 Elsevier B.V. All rights reserved

ObjectiveSudden cardiac death (SCD) in families with premature atherosclerosis (PAS) is generally attributed to lethal arrhythmias during myocardial infarction. Yet, such arrhythmias may also arise from non-ischaemic inherited susceptibility. We aimed to test the hypothesis that Brugada syndrome is prevalent among families with PAS in which SCD occurred.MethodsWe investigated all patients who underwent Ajmaline testing to screen them for Brugada syndrome because of unexplained familial SCD in the Amsterdam University Medical Centers between 2004 and 2017. We divided the cohort into two groups based on a positive family history for PAS. All individuals with a positive Ajmaline test were screened for SCN5A-mutation.ResultsIn families with SCD and PAS, the prevalence of positive Ajmaline test was similar to families with SCD alone (22% vs 19%). The number of SCD cases in families with SCD and PAS was higher (2.34 vs 1.63, pConclusionsBrugada syndrome has a similar prevalence in families with SCD and PAS as in families with SCD alone, although SCD in families with SCD and PAS occurs in more family members and at older age, while SCN5A mutations in these families are rare. This suggests that the SCD occurring in families with PAS could be related to an underlying genetic predisposition of arrhythmias, with a different genetic origin. It could be considered to screen families with SCD and PAS for Brugada syndrome.

Background Damage to endothelial glycocalyx is thought to be an early marker of atherosclerosis and measuring reduced glycocalyx size clinically via the Perfused Boundary Region (PBR) may allow early detection of cardiovascular disease. However, the true value of the glycocalyx in estimating cardiovascular risk or detecting cardiovascular disease is uncertain. We therefore investigated whether small glycocalyx size is associated with cardiovascular risk or disease in a large multi-ethnic cohort. Methods In a multi-ethnic community-based sample (N = 6169, 42.4% male, mean age 43.6 ±13) we applied multiple imputation for missing data and used logistic regression and odds ratios to cross-sectionally investigate the relationship of small glycocalyx size as estimated by highest quartile of PBR with, on the one hand, classical risk factors for atherosclerosis including age, sex, diastolic and systolic blood pressure, LDL, HDL, triglycerides, BMI, diabetes, smoking status, and antihypertensive and lipid-lowering medication; on the other hand, prevalent cardiovascular disease. Analyses were additionally adjusted for ethnicity. Results With PBR divided in quartiles, the highest PBR quartile (smallest glycocalyx size) as dependent variable was independently associated with female sex (OR for male versus female: 0.61, 95% CI: 0.53, 0.70) and diabetes (OR: 1.28, 95% CI: 1.03-1.59) in a model adjusted for all classical risk factors of atherosclerosis and for ethnicity. With regard to cardiovascular disease, no association was found between the smallest glycocalyx size as independent variable and overall cardiovascular disease, coronary heart disease and revascularization procedures, or stroke. Conclusions Small glycocalyx size as estimated by highest PBR is associated with female sex and diabetes, which do not completely reflect a high cardiovascular risk profile. At the same time, glycocalyx size is not associated with prevalent cardiovascular disease.

BackgroundChanges in the microcirculation may be used as a surrogate outcome in studies on cardiovascular disease. We assessed the reliability characteristics of the sublingual microcirculation parameters Vascular Density (VD), Red Blood Cell Filling (RBCF), and Perfused Boundary Region (PBR) as obtained by sidestream darkfield imaging.MethodsFor each of the three parameters, the variance components of measurement, the Intraclass Correlation Coefficient (ICC), the Standard Error of Measurement, and the limits of agreement were estimated for the intra-rater setting (N = 50) and the inter-rater setting (N = 48). Subsequently, as a proof of concept, the reliability measures were used for a power analysis to design studies to evaluate the effect of acute stimuli-i.e. having a meal (N = 50) and cigarette smoking (N = 21) on the three parameters.ResultsReproducibility was poor for all three parameters. The intra-rater ICC for 2 measurements was 0.28 (95% CI: 0.04, 0.53) for the VD, 0.51 (95% CI: 0.27, 0.69) for the RBCF, and 0.33 (95% CI: 0.08-0.56) for the PBR. The standard errors of measurement and the limits of agreement for all three parameters were larger than most statistically significant intra-individual or inter-individual differences reported in previous studies. The proofs of concept showed that sample sizes in excess of 600 subjects are necessary to reach statistical significance for the observed effects of having a meal or smoking on VD and PBR.ConclusionsThe reliability of the three sublingual microcirculation parameters in their current form appears to be low and a large sample size is advisable for their use in conditions similar to those we describe.

Trillions of microorganisms inhabit the human gut and are regarded as potential key factors for health

Trillions of microorganisms inhabit the human gut and are regarded as potential key factors for health1,2. Characteristics such as diet, lifestyle, or genetics can shape the composition of the gut microbiota2–6 and are usually shared by individuals from comparable ethnic origin. So far, most studies assessing how ethnicity relates to the intestinal microbiota compared small groups living at separate geographical locations7–10. Using fecal 16S ribosomal RNA gene sequencing in 2,084 participants of the Healthy Life in an Urban Setting (HELIUS) study11,12, we show that individuals living in the same city tend to share similar gut microbiota characteristics with others of their ethnic background. Ethnicity contributed to explain the interindividual dissimilarities in gut microbiota composition, with three main poles primarily characterized by operational taxonomic units (OTUs) classified as Prevotella (Moroccans, Turks, Ghanaians), Bacteroides (African Surinamese, South-Asian Surinamese), and Clostridiales (Dutch). The Dutch exhibited the greatest gut microbiota α-diversity and the South-Asian Surinamese the smallest, with corresponding enrichment or depletion in numerous OTUs. Ethnic differences in α-diversity and interindividual dissimilarities were independent of metabolic health and only partly explained by ethnic-related characteristics including sociodemographic, lifestyle, or diet factors. Hence, the ethnic origin of individuals may be an important factor to consider in microbiome research and its potential future applications in ethnic-diverse societies.

Since the discovery of microRNAs (miRNAs), circulating miRNAs have been proposed as biomarkers for disease. Consequently, many groups have tried to identify circulating miRNA biomarkers for various types of diseases including cardiovascular disease and cancer. However, the replicability of these experiments has been disappointingly low. In order to identify circulating miRNA candidate biomarkers, in general, first an unbiased high-throughput screen is performed in which a large number of miRNAs is detected and quantified in the circulation. Because these are costly experiments, many of such studies have been performed using a low number of study subjects (small sample size). Due to lack of power in small sample size experiments, true effects are often missed and many of the detected effects are wrong. Therefore, it is important to have a good estimate of the appropriate sample size for a miRNA high-throughput screen. In this review, we discuss the effects of small sample sizes in high-throughput screens for circulating miRNAs. Using data from a miRNA high-throughput experiment on isolated monocytes, we illustrate that the implementation of power calculations in a high-throughput miRNA discovery experiment will avoid unnecessarily large and expensive experiments, while still having enough power to be able to detect clinically important differences. Keywords: MicroRNA, High-throughput screens, Biomarkers, Small sample size error, Array, Methodology

Small studies suggested circulating microRNAs (miRNAs) as biomarkers for heart failure (HF). However, standardized approaches and quality assessment for measuring circulating miRNAs are not uniformly established, and most studies have been small, so that results are inconsistent. We used a standardized data handling protocol, optimized for circulating miRNA qPCRs to remove noise and used it to assess which circulating miRNAs robustly add prognostic information in patients with HF.We measured 12 miRNAs in two independent cohorts totalling 2203 subjects. Cohort I (Barcelona) comprised 834 chronic HF patients. Cohort II (Detroit) comprised 1369 chronic HF patients. Each sample was measured in duplicate, and normalized to a very abundant and stable miRNA (miR-486-5p). We used a multistep algorithm to distinguish false amplification signals and thus classify each miRNA measurement as 'valid', 'undetectable' or 'invalid'. Higher levels of miR-1254 and miR-1306-5p were significantly associated with risk of the combined endpoint of all-cause mortality and HF hospitalization in both cohorts, with hazard ratios ranging from 1.11 to 1.21 per log increase (P-values 0.004 to 0.009). However, adding these miRNAs to established predictors (age, sex, haemoglobin, renal function, and NT-proBNP) did not further augment the c-statistic beyond 0.69 (cohort I) or 0.70 (cohort II).We used a stringent quality assessment for miRNA testing, and were able to replicate the association of miR-1254 and miR-1306-5p with risk of death and HF hospitalization in HF patients of two independent cohorts. However, these two circulating miRNAs failed to improve prognostication over established predictors.

BackgroundLamin A/C (LMNA) mutation carriers suffer from a variety of clinical phenotypes, including dilated cardiomyopathy (DCM). Although it has been suggested that carriers are at risk for thromboembolic complications, it is unknown whether this risk is higher than can be expected from the underlying cardiac abnormalities. The purpose of this study was to determine whether a LMNA mutation is associated with an increased risk of thromboembolic complications.MethodsWe compared a cohort of 76 LMNA mutation carriers with a cohort of 224 idiopathic DCM patients without a LMNA mutation, with respect to the prevalence of arterial and venous thromboembolic complications. Furthermore, we carried out a case–control study to explore whether a prothrombotic phenotype was present in LMNA mutation carriers without DCM or atrial tachyarrhythmias (n=14) and compared this with mutation negative relatives (n=13).ResultsThe prevalence of thromboembolic complications was higher in the cohort of LMNA mutation carriers than in DCM patients (22 vs 11%; p

Stratification for subsequent coronary events among patients with coronary artery disease (CAD) is of considerable interest because of the potential to guide secondary preventive therapies. Recently, we identified eight microRNAs (miRNAs), which facilitated acute coronary syndrome (ACS) diagnosis. In this study, we aimed to evaluate their potential role as prognostic biomarkers for cardiovascular disease.The serum concentrations of eight candidate miRNAs -miR-19a, miR-19b, miR-132, miR-140-3p, miR-142-5p, miR-150, miR-186, and miR-210 were measured in a cohort of 1112 patients with documented CAD-including 430 patients with ACS and 682 patients with stable angina pectoris. Cardiovascular death was the main outcome measure.During a median follow-up of 4.0 years, most miRNAs reliably predicted cardiovascular death in ACS patients. Cox regression analyses indicated that in particular miR-132 (HR 2.85 per 1 SD increase, P = 0.022), miR-140-3p (HR 2.88 per 1 SD increase, P = 0.022), and miR-210 (HR 3.10 per 1 SD increase, P = 0.039) were able to precisely predict cardiovascular death. Circulating miR-132, miR-140-3p, and miR-210 clearly improved various model performance measures, including C-statistics (AUC [area under the receiver-operating characteristic curve] for miR-132: 0.737; AUC for miR-140-3p: 0.756; AUC for miR-210: 0.754).This is the largest study so far evaluating the prognostic value of circulating miRNAs in cardiovascular disease. Our study shows that single miRNAs derived from peripheral blood predict mortality in secondary prevention settings, and thereby represent valuable biomarkers for risk estimation in CAD.

Background Hypertension alone is a poor predictor of the individual risk of cardiovascular disease. Hereditary factors of which hypertension is merely a marker may explain why some hypertensive individuals appear more susceptible to cardiovascular disease, and why some ethnicities have more often seemingly hypertension‐related cardiovascular disease than others. We hypothesize that, in hypertensive individuals, a positive family history of cardiovascular disease identifies a high‐risk subpopulation. Methods and Results Healthy Life in Urban Settings (HELIUS) is a cohort study among participants of Dutch, South‐Asian Surinamese, African Surinamese, Ghanaian, Turkish, and Moroccan origin aged 70 years and younger. In participants with hypertension (n=6467), we used logistic regression to assess the association of family history of cardiovascular disease with prevalent stroke and nonstroke cardiovascular disease, adjusting for sex, age, education, and smoking. To detect ethnic differences, we tested for interaction between family history and ethnicity and stratified the analysis by ethnicity. A positive family history was associated with a higher prevalence of nonstroke cardiovascular disease (odds ratio [OR], 2.05; 95% CI , 1.65–2.54) and stroke (OR, 1.62; 95% CI , 1.19–2.20). The strongest association of family history with nonstroke cardiovascular disease was found among the Dutch (OR, 2.47; 95% CI, 1.37–4.44) and with stroke among the African Surinamese (OR, 2.17; 95% CI, 1.32–3.57). The interaction between family history and African Surinamese origin for stroke was statistically significant. Conclusions In multiethnic populations of hypertensive patients, a positive family history of cardiovascular disease may be used clinically to identify individuals at high risk for nonstroke cardiovascular disease regardless of ethnic origin and African Surinamese individuals at high risk for stroke.

Objectives The goal of this study was to evaluate whether individuals with a positive family history for premature coronary artery disease (CAD) and coronary calcium scoring (CCS) above the 80th percentile might benefit from preventive treatment. Background First-degree relatives of patients with premature CAD have an increased risk for cardiovascular disease (CVD), whereas events are poorly predicted in these individuals. Surrogate markers, such as CCS, might refine risk scoring. Nevertheless, the outcome of the St. Francis Heart trial, which investigated the effect of atorvastatin 20 mg/day in asymptomatic individuals with CCS above the 80th percentile, did not reach statistical significance. Methods We performed a post hoc analysis on the database of the St. Francis trial to assess efficacy of treatment with atorvastatin 20 mg/day in those with CCS above the 80th percentile and presence (n = 543) or absence (n = 462) of a positive family history for premature CAD. All participants received aspirin 81 mg/day. Primary outcome included coronary death, myocardial infarction, coronary revascularization, stroke, and arterial surgery. Results A total of 1,005 individuals, with a mean age of 59.0 ± 5.9 years and a median absolute CCS of 370 Agatston units (interquartile range: 183 to 662) participated in the trial. After a follow-up of 4.3 (interquartile range: 3.5 to 4.5) years, 7.2% of the treated individuals with a positive family history had a cardiovascular event versus 12.5% of the placebo group (hazard ratio [HR]: 0.55; 95% confidence intervals [CI]: 0.31 to 0.97; p = 0.040). This is comparable with a number needed to treat of 18.9. In individuals without a family history, events were minimally reduced: 6.6% in the treated versus 6.8% in the placebo group (HR: 1.04; 95% CI: 0.51 to 2.13; p = 0.912). Conclusions The combination of a positive family history and CCS above the 80th percentile identifies a subgroup within the primary prevention population that receives greater benefit from statin treatment than the population at large. These results have important implications for future guidelines concerning individuals with a positive family history for premature CAD.

Background: Cardiovascular disease (CVD) is characterized by slow progressive atherosclerosis and arterial thrombotic events, leading to occlusions. Whether either of these presentations is more likely in patients with a genetic predisposition for CVD is still unknown. We suggest that a genetic predisposition for CVD is related to recurrent events of the same nature. Methods: We retrospectively investigated 275 patients with premature CVD and divided them in two groups according to their first event: an arterial thrombotic event or stable atherosclerosis. We used a Cox proportional-hazards model to estimate the effect of a positive family history for CVD on recurrent events of the same nature. This was tested in the entire cohort and in patients with coronary artery disease only. Results: Patients with a first arterial thrombotic event and a positive family history had a threefold increased risk for a recurrent event of the same nature, compared to patients with a negative family history (hazard ratio 3.00, 95% confidence interval 1.32-6.81); p

Background The prognosis of patients with haematological malignancies who are admitted to the ICU is generally poor. In order to optimize care, it is important to be able to determine which patients are most likely to benefit from continuation of treatment after ICU admission. Methods Data of 86 patients with a haematological malignancy consecutively admitted to the ICU of Maastricht University Medical Centre were examined in a retrospective cohort study in order to identify clinically useful prognostic parameters. Results ICU mortality was 56% and in-hospital mortality was 65%. Non-survivors had higher APACHE-II and SOFA scores compared with survivors (32 ± 8.0 versus 25 ± 6.5 and 11.5 ± 3.1 versus 8.5 ± 3.0, respectively). The mortality rate was significantly higher in patients with an increasing SOFA score (≥ 2 points) compared with patients with an unchanged or decreasing SOFA score (72% versus 58% and 21%, respectively). Mortality was also higher in patients requiring invasive mechanical ventilation or inotropic/vasopressor therapy. Conclusion The mortality rate among patients with haematological malignancies who are admitted to the ICU is high and mainly associated with the severity of illness, as reflected by more severe and worsening organ failure and a need for mechanical ventilation or inotropic/vasopressor therapy. Several factors appear to be associated with a poor outcome, but no absolute predictors of mortality could be identified, although the results suggest that changes in the SOFA score during the stay in the ICU can be helpful in the decision making about the continuation or discontinuation of treatment.

Background: Premature cardiovascular disease (CVD) is treated in the same way as CVD of advanced age. However, in patients with premature CVD and a family history of CVD, different -possibly genetic-mechanisms may underlie this disease, which current medical treatment is not targeted to. This suggests that subjects with a genetic predisposition to CVD are more likely to have recurrent cardiovascular events. Methods: We retrospectively investigated 291 patients with premature CVD and assessed the amount of recurrent events according to family history in a follow-up period of 31 years. Premature CVD was defined as an event

The 15th Annual Meeting of the European Council of Cardiovascular Research brought together basic and clinical scientists working in the cardiovascular field in La Colle sur Loup, France. Upfront basic and clinical research addressing the mechanisms of disease, identification of biomarkers or development of new treatments was communicated in 101 presentations, 35 of them as a part of five on-topic oral sessions and three workshops. Three keynote lectures reviewed current knowledge and the latest data about mechanosensitive channels in pressure regulation, cell therapy in cardiovascular disease and mechanisms of cardiovascular risk associated with diabetic nephropathy. This article summarizes highlights of the oral sessions, workshops and keynote lectures.

Background Elevated lipoprotein(a) (Lp(a)) levels are associated with increased risk for atherosclerotic cardiovascular disease (ASCVD). Individuals with a family history of premature ASCVD are at increased cardiovascular risk with concomitantly a higher burden of (subclinical) atherosclerosis. However, whether Lp(a) contributes to the increased atherosclerotic burden in these individuals remains to be established. Objective In this study, we evaluated the association between Lp(a) levels and coronary atherosclerotic burden, assessed by coronary arterty calcium (CAC) scores, in asymptomatic individuals with a family history of premature ASCVD. Methods Lp(a) levels and other ASCVD risk factors were assessed in 432 individuals with premature ASCVD and in 937 healthy asymptomatic family members. CAC scores were only measured in asymptomatic family members. Results In this cohort, 16% had elevated Lp(a) levels, defined as ≥ 50 mg/dL. Among healthy family members, elevated Lp(a) levels were associated with both absolute CAC scores of ≥ 100 (odds ratio [OR] 1.79 [95% confidence interval {CI} 1.13–2.83]) as well as with age- and gender-corrected CAC scores ≥ 80th percentile (OR 1.69 [95% CI 1.14–2.50]). This coincides with a higher prevalence of cardiovascular events (OR 1.48 [95% CI 1.11–2.01]) in the whole cohort. Conclusion Elevated Lp(a) levels were associated with higher CAC scores, both absolute as well as age- and gender-corrected percentiles, in individuals with a family history of premature ASCVD. These data imply that Lp(a) accelerates progression of atherosclerosis in these individuals, thereby contributing to their increased ASCVD risk.

BACKGROUND: Coronary collateral arteries function as natural bypasses in the event of coronary obstruction. The degree of collateral network development significantly impacts the outcome of patients after an acute myocardial infarction (AMI). MicroRNAs (miRNAs, miRs) have arisen as biomarkers to identify heterogeneous patients, as well as new therapeutic targets in cardiovascular disease. We sought to identify miRNAs that are differentially expressed in chronic total occlusion (CTO) patients with well or poorly developed collateral arteries. METHODS AND RESULTS: Forty-one CTO patients undergoing coronary angiography and invasive assessment of their coronary collateralization were dichotomized based on their collateral flow index (CFI). After miRNA profiling was conducted on aortic plasma, four miRNAs were selected for validation by real-time quantitative reverse transcription polymerase chain reaction in patients with low (CFI0.39) collateral artery capacity. We confirmed significantly elevated levels of miR423-5p (p

Circulating microRNAs (miRNAs) have been reported as biomarkers for disease diagnosis. RT- qPCR is most commonly used to detect miRNAs; how- ever, no consensus on the most appropriate method for data normalization exists. Via a standardized selection method, we aimed to determine separate miRNA nor- malization panels for RT-qPCR measurements on whole blood, platelets, and serum. Candidate miRNAs were selected from studies describing circulating miRNA microarray data in the Gene Expression Omnibus or ArrayExpress. miRNA expression data of healthy controlswereretrievedfromeachstudy.Foreachsample type, we selected those miRNAs that were least variable and sufficiently highly expressed in multiple microarray experiments, performed on at least 2 different plat- forms. Stability of the candidate miRNAs was assessed using NormFinder and geNorm algorithms in a RT- qPCR cohort of 10 patients with coronary artery disease and 10 healthy controls. We selected miRNA normaliza- tion panels forRT-qPCR measurements onwholeblood, platelets, and serum. As a validation, we assessed the precision of all 3 panels in 3 independent RT-qPCR cohorts and compared this with normalization for miR-16 or RNU6B. The proposed normalization panels for whole blood, platelets, and serum show better pre- cision than normalization for miR-16 or RNU6B.—Kok, M. G. M., Halliani, A., Moerland, P. D., Meijers, J. C. M., Creemers, E. E., Pinto-Sietsma, S.-J. Normal- ization panels for the reliable quantification of circulating microRNAs by RT-qPCR. FASEB J. 29, 000-000 (2015). www.fasebj.org

Previous studies have shown that low birth weight (LBW) is a risk factor for renal impairment in adult life. The effects of LBW and renal function were studied by using twins, which allows distinguishing among fetoplacental, maternal, and genetic influences. Perinatal data were obtained at birth, and absolute creatinine clearance (not corrected for body surface area) was measured at a mean age of 25.6 yr in 653 individuals. Twins were considered both as individuals and as members of twin pairs. Statistical analyses were performed with and without adjusting for gestational age, zygosity, gender, age, body mass index, glucose level, BP, and smoking status. Creatinine clearance was 4 ml/min lower in twins with LBW (

a Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands b Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands c Department of Hypertension and Dyslipidemia, Geriatric Clinic and European Center on Atherosclerosis, G. D'Annunzio University, Chieti, Italy d Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands e Department of Experimental Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands f Department of Clinical Chemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands g Department of Plasma Proteins, Sanquin, Amsterdam, The Netherlands

A decreased GFR in the range of mild renal insufficiency and an increased urinary albumin excretion (UAE) rate in the range of microalbuminuria are important cardiovascular risk factors. Endothelin-1 (ET-1) has been suggested to be a major disease promoting factor in renal disease. The role of the ET-1 gene locus (EDN1) for renal function in the general nondiabetic population was evaluated. To explore the overall relevance of EDN1, two suitable single-nucleotide polymorphisms, EDN1 K198N and EDN1 T-1370G, were selected, and haplotype analysis was performed. Determined were genotypes in 7291 nondiabetic subjects from the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study. Genetic analysis was related to UAE and GFR as continuous variables and to microalbuminuria and diminished filtration as dichotomous traits. In a logistic regression analysis, no significant higher risk for increased UAE, microalbuminuria, decreased GFR, or diminished filtration could be observed for either single-nucleotide polymorphism separately. Haplotype analysis revealed that individuals with the homozygous G-N haplotype (compound EDN1 - 1370GG/198NN genotype) have a lower GFR than the remaining subjects (P

Abstract —Angiotensin II recruits transforming growth factor β 1 (TGFβ 1 ) and is related to left ventricular fibrosis. However, it is unclear whether chronic in vivo reduction in left ventricular TGFβ 1 expression blunts fibrosis and improves outcome in angiotensin II–dependent hypertension. Four-week-old male hypertensive TGR(mRen2)27 (Ren2) rats received either normal food, low-dose losartan (0.5 mg · kg −1 · d −1 ), or tranilast (a nonspecific TGFβ inhibitor; 400 mg · kg −1 · d −1 ) (n=10 for each group) for 12 weeks and were compared with Sprague-Dawley control rats. The effect of tranilast on survival was evaluated in 34 additional untreated homozygous Ren2 rats. Tranilast or low-dose losartan did not lower blood pressure. However, the increase in left ventricular weight (Ren2 versus SD 3.1±0.16 versus 2.1± 0.06 mg/g body wt; P P 1 mRNA and fibronectin mRNA and blunted the increase in hydroxyproline content and the increase in perivascular fibrosis. The perivascular fibrosis score correlated significantly with the level of expression of TGFβ 1 ( r =0.62; P =0.019). In situ hybridization demonstrated increases in TGFβ 1 mRNA, predominantly in perivascular and nonmyocyte areas. Both drugs did not prevent the decrease in systolic or diastolic dP/dt, but tranilast significantly improved the survival of untreated Ren2 rats ( P =0.029). In conclusion, TGFβ 1 mRNA expression is increased predominantly in nonmyocyte regions in the hypertrophied left ventricle in this angiotensin II–dependent model of hypertension. This increase is probably due to high angiotensin II levels rather than to hypertension. This is the first study to suggest that chronic inhibition of TGFβ 1 expression attenuates left ventricular hypertrophy and fibrosis, even without lowering blood pressure.

A role for endothelin in the pathogenesis of hypertension: Fact or fiction? Endothelin-1 (ET-1) was discovered 10years ago. Because it is one of the most potent vasoconstrictors in vivo, a pathophysiological role for the peptide as a mediator of hypertension has been postulated. Several clinical studies, however, have been unable to identify elevated ET levels in the plasma of hypertensive patients, suggesting that it does not play a prominent role in this disease. More recently, evidence has been presented that ETs act predominantly at the autocrine/paracrine level and that measurements of plasma levels can give only an indirect view of the activity of the system. In addition, transgenic technology has uncovered new actions of the peptide systems in recent years, which point to a key function of the system in prenatal development. Moreover, investigation of conditions associated with hypertensive end-organ damage, such as chronic renal failure, has led to a re-evaluation of the role of the ET system in hypertension. This article discusses this recent evidence and defines the exact role of the ET system in hypertension and hypertensive end-organ damage.