Platelets in Patients with Premature Coronary Artery Disease Exhibit Upregulation of miRNA340* and miRNA624*

Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. It remains unknown whether differences in miRNA expression levels in platelets can be found between patients with premature CAD and healthy controls. In this case-control study we measured relative expression levels of platelet miRNAs using microarrays from 12 patients with premature CAD and 12 age- and sex-matched healthy controls. Six platelet microRNAs were significantly upregulated (miR340*, miR451, miR454*, miR545:9.1. miR615-5p and miR624*) and one miRNA (miR1280) was significantly downregulated in patients with CAD as compared to healthy controls. To validate these results, we measured the expression levels of these candidate miRNAs by qRT-PCR in platelets of individuals from two independent cohorts; validation cohort I consisted of 40 patients with premature CAD and 40 healthy controls and validation cohort II consisted of 27 patients with artery disease and 40 healthy relatives. MiR340* and miR624* were confirmed to be upregulated in patients with CAD as compared to healthy controls in both validation cohorts. Two miRNAs in platelets are significantly upregulated in patients with CAD as compared to healthy controls. Whether the two identified miRNAs can be used as biomarkers and whether they are cause or consequence of the disease remains to be elucidated in a larger prospective stud

A pooled-analysis of age and sex based coronary artery calcium scores percentiles

Funding The authors of this publication did not receive any grant from funding agencies in the public, commercial, or not-for-profit sector to support this research effort. Dr. Paolo Raggi was supported by a grant (RES0016825) from the Faculty of Medicine and Dentistry at the University of Alberta, Edmonton, AB, Canada. Dr. Matthew J. Budoff has Grant support from general electric and NIH. None of the other authors declares a conflict of interest. Publisher Copyright: © 2020 [The Author/The Authors]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.Peer reviewe

Rationale and design of the PRAETORIAN-COVID trial:A double-blind, placebo-controlled randomized clinical trial with valsartan for PRevention of Acute rEspiraTORy dIstress syndrome in hospitAlized patieNts with SARS-COV-2 Infection Disease

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). 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

Monocyte Gene Expression Signature of Patients with Early Onset Coronary Artery Disease

The burden of cardiovascular disease (CVD) cannot be fully addressed by therapy targeting known pathophysiological pathways. Even with stringent control of all risk factors CVD events are only diminished by half. A number of additional pathways probably play a role in the development of CVD and might serve as novel therapeutic targets. Genome wide expression studies represent a powerful tool to identify such novel pathways. We compared the expression profiles in monocytes from twenty two young male patients with premature familial CAD with those from controls matched for age, sex and smoking status, without a family history of CVD. Since all patients were on statins and aspirin treatment, potentially affecting the expression of genes in monocytes, twelve controls were subsequently treated with simvastatin and aspirin for 6 and 2 weeks, respectively

Association of Family History With Cardiovascular Disease in Hypertensive Individuals in a Multiethnic Population

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. 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. 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 strok

Reproducibility of sublingual microcirculation parameters obtained from sidestream darkfield imaging

BACKGROUND: Changes 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. METHODS: For 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. RESULTS: Reproducibility 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. CONCLUSIONS: The 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

Sublingual endothelial glycocalyx and atherosclerosis. A cross-sectional study.

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

Reproducibility of sublingual microcirculation parameters obtained from sidestream darkfield imaging.

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

Study design and qPCR data analysis guidelines for reliable circulating miRNA biomarker experiments: A review

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