Maurice W.J. de Ronde

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

Background 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. Methodology/Principal Findings 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. Conclusion/Significance 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 study.

Prognostic value of circulating microRNAs on heart failure-related morbidity and mortality in two large diverse cohorts of general heart failure patients

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.

Circulating MicroRNAs Characterizing Patients with Insufficient Coronary Collateral Artery Function.

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 (CFI<0.39) and high (CFI>0.39) collateral artery capacity. We confirmed significantly elevated levels of miR423-5p (p<0.05), miR10b (p<0.05), miR30d (p<0.05) and miR126 (p<0.001) in patients with insufficient collateral network development. We further demonstrated that each of these miRNAs could serve as circulating biomarkers to discriminate patients with low collateral capacity (p<0.01 for each miRNA). We also determined significantly greater expression of miR30d (p<0.05) and miR126 (p<0.001) in CTO patients relative to healthy controls. Conclusion The present study identifies differentially expressed miRNAs in patients with high versus low coronary collateral capacity. We have shown that these miRNAs can function as circulating biomarkers to discriminate between patients with insufficient or sufficient collateralization. This is the first study to identify miRNAs linked to coronary collateral vessel function in humans.

Serially measured circulating microRNAs and adverse clinical outcomes in patients with acute heart failure.

Previous studies have identified candidate circulating microRNAs (circmiRs) as biomarkers for heart failure (HF) using relatively insensitive arrays, validated in small cohorts. The present study used RNA sequencing to identify novel candidate circmiRs and compared these with previously identified circmiRs in a large, prospective cohort of patients with acute HF (AHF).

Reporting Weaknesses in Conference Abstracts of Diagnostic Accuracy Studies in Ophthalmology.

IMPORTANCE Conference abstracts present information that helps clinicians and researchers to decide whether to attend a presentation. They also provide a source of unpublished research that could potentially be included in systematic reviews. We systematically assessed whether conference abstracts of studies that evaluated the accuracy of a diagnostic test were sufficiently informative. OBSERVATIONS We identified all abstracts describing work presented at the 2010 Annual Meeting of the Association for Research in Vision and Ophthalmology. Abstracts were eligible if they included a measure of diagnostic accuracy, such as sensitivity, specificity, or likelihood ratios. Two independent reviewers evaluated each abstract using a list of 21 items, selected from published guidance for adequate reporting. A total of 126 of 6310 abstracts presented were eligible. Only a minority reported inclusion criteria (5%), clinical setting (24%), patient sampling (10%), reference standard (48%), whether test readers were masked (7%), 2 × 2 tables (16%), and confidence intervals around accuracy estimates (16%). The mean number of items reported was 8.9 of 21 (SD, 2.1; range, 4-17). CONCLUSIONS AND RELEVANCE Crucial information about study methods and results is often missing in abstracts of diagnostic studies presented at the Association for Research in Vision and Ophthalmology Annual Meeting, making it difficult to assess risk for bias and applicability to specific clinical settings.

Circulating microRNA biomarkers for cardiovascular risk prediction: are we approaching clinical application?

Patients at high risk of cardiovascular morbidity and mortality are still difficult to identify. Unfortunately, the most widely used standard risk prediction models predict cardiovascular disease (CVD) rather poorly, since these risk calculators are mostly driven by age. Therefore, most young subjects are regarded as low risk merely based on their age, despite sometimes quite obvious adverse risk factors. Circulating biomarkers that can help to improve the identification of individuals at risk are therefore highly needed.

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.