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

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

Reporting Weaknesses in Conference Abstracts of Diagnostic Accuracy Studies in Ophthalmology

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. 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). 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 setting

Elevated lipoprotein(a) levels are associated with coronary artery calcium scores in asymptomatic individuals with a family history of premature atherosclerotic cardiovascular disease

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

MiR-223-3p and miR-122-5p as circulating biomarkers for plaque instability

BACKGROUND: In this study, we discovered and validated candidate microRNA (miRNA) biomarkers for coronary artery disease (CAD). METHOD: Candidate tissue-derived miRNAs from atherosclerotic plaque material in patients with stable coronary artery disease (SCAD) (n=14) and unstable coronary artery disease (UCAD) (n=25) were discovered by qPCR-based arrays. We validated differentially expressed miRNAs, along with seven promising CAD-associated miRNAs from the literature, in the serum of two large cohorts (n=395 and n=1000) of patients with SCAD and UCAD and subclinical atherosclerosis (SubA) and controls, respectively. RESULT: From plaque materials (discovery phase), miR-125b-5p and miR-193b-3p were most upregulated in SCAD, whereas miR-223-3p and miR-142-3p were most upregulated in patients with UCAD. Subsequent validation in serum from patients with UCAD, SCAD, SubA and controls demonstrated significant upregulation of miR-223-3p, miR-133a-3p, miR-146-3p and miR-155-5p. The ischaemia-related miR-499-5p was also highly upregulated in patients with UCAD compared with the other groups (SCAD OR 20.63 (95% CI 11.16 to 38.15), SubA OR 96.10 (95% CI 40.13 to 230.14) and controls OR 15.73 (95% CI 7.80 to 31.72)). However, no significant difference in miR-499-5p expression was observed across SCAD, SubA and controls. MiR-122-5p was the only miRNA to be significantly upregulated in the serum of both patients with UCAD and SCAD. CONCLUSION: In conclusion, miR-122-5p and miR-223-3p might be markers of plaque instability

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

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

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 (CFI0.39) 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

High miR-124-3p expression identifies smoking individuals susceptible to atherosclerosis

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 1.05-7.01). 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

High miR-124-3p expression identifies smoking individuals susceptible to atherosclerosis

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 reserve