Pioneering studies have revealed the presence of endogenous microRNAs (miRNAs) in the circulation that are not cell-associated. 1 The cellular origin and the biological function of circulating miRNAs, however, are less clear. Editorial, see p 576We have previously quantified circulating miRNAs in a large population-based cohort, the Bruneck study.3,4 Using concepts of network topology, 5 we identified altered miRNA signatures in patients with type 2 diabetes mellitus 3 and with future myocardial infarction. 4 In addition, we subjected healthy volunteers to limb ischemia-reperfusion generated by thigh cuff inflation. 4 Computational analysis identified 6 distinct miRNA clusters. 4 One cluster included all miRNAs associated with risk of myocardial infarction and consisted of miRNAs predominantly expressed in platelets. Microarray screening revealed that miR-126, miR-197, miR-223, miR-24, and miR-21 are among the most highly expressed miRNAs in platelets and platelet microparticles (PMPs), and their circulating levels correlated with PMPs as quantified by flow cytometry.Original received November 14, 2012; revision received December 21, 2012; accepted December 28, 2012. In November 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.8 days.Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope.

M icroRNAs (miRNAs) are small noncoding RNAs with cell-type specific expression patterns that are released by cells into the circulation as part of membranous particles or protein complexes.1 Thus, miRNAs can be readily quantified by real-time polymerase chain reactions (qPCRs) in plasma and serum and have generated increasing interest as potential new biomarkers.2 Our group has previously identified plateletrelated miRNA signatures that are predictive of cardiovascular events. 3 In addition, we measured miRNAs in healthy volunteers and in patients with symptomatic atherosclerosis before and after initiation of dual antiplatelet therapy and demonstrated reduced plasma levels of platelet-related miRNAs on platelet inhibition. Kaudewitz et al Plasma MicroRNAs and Platelet Function 421Dual oral antiplatelet therapy (acetylsalicylic acid [ASA]+a P2Y 12 inhibitor) is commonly used for the management of non-ST-segment-elevation acute coronary syndromes (ACS) and ST-segment-elevation myocardial infarction.5 ASA irreversibly inhibits cyclooxygenase 1 in platelets, thereby repressing thromboxane A 2 (TxA 2 ) synthesis and, consequently, platelet activation. Clopidogrel, prasugrel, and ticagrelor target the P2Y 12 receptor for ADP. However, interindividual variability in the platelet response to clopidogrel has been reported. Prasugrel and ticagrelor exhibit a more consistent antiplatelet effect and have shown benefits over clopidogrel in patients with ACS but also increase the risk of bleeding. 6,7 It is currently unclear whether plasma levels of platelet-related miRNAs correlate with the residual platelet activity in patients with ACS and how different antiplatelet agents alter miRNAs.In this study, we used RNA sequencing to characterize small RNAs in plasma. Then, we compared the effect of different antiplatelet agents and explored the association of small RNAs (miRNAs and YRNAs) with platelet function tests in patients with ACS. Moreover, we correlated their plasma levels to platelet activation markers in the prospective, population-based Bruneck study 3 and investigated whether a single-nucleotide polymorphism (SNP) that facilitates miR-126 processing 8 alters circulating miR-126 levels and platelet reactivity. These epidemiological observations were complemented by preclinical studies, assessing platelet function in mice on treatment with antagomiRs directed against miR-126 and by mechanistic studies measuring miR-126 targets. MethodsAn expanded Methods section is available in the Online Data Supplement. Next-Generation SequencingSmall RNA libraries were generated from non-normalized RNA (ranging from 375 pg to 1 ng) extracted from equal volumes of platelet-poor plasma (PPP) and platelet-rich plasma (PRP) from healthy human volunteers. Before library preparation, RNA was spiked with equal amounts of C. elegans miR-39 star (cel-miR-39*) to assist in normalization. Libraries were prepared using the small RNA library preparation kit version 2.0 (Illumina Cambridge Ltd) according to manufacturer's protocol with limi...

MicroRNA-122 (miR-122) is abundant in the liver and involved in lipid homeostasis, but its relevance to the long-term risk of developing metabolic disorders is unknown. We therefore measured circulating miR-122 in the prospective population-based Bruneck Study (n = 810; survey year 1995). Circulating miR-122 was associated with prevalent insulin resistance, obesity, metabolic syndrome, type 2 diabetes, and an adverse lipid profile. Among 92 plasma proteins and 135 lipid subspecies quantified with mass spectrometry, it correlated inversely with zinc-α-2-glycoprotein and positively with afamin, complement factor H, VLDL-associated apolipoproteins, and lipid subspecies containing monounsaturated and saturated fatty acids. Proteomics analysis of livers from antagomiR-122–treated mice revealed novel regulators of hepatic lipid metabolism that are responsive to miR-122 inhibition. In the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT, n = 155), 12-month atorvastatin reduced circulating miR-122. A similar response to atorvastatin was observed in mice and cultured murine hepatocytes. Over up to 15 years of follow-up in the Bruneck Study, multivariable adjusted risk ratios per one-SD higher log miR-122 were 1.60 (95% CI 1.30–1.96; P < 0.001) for metabolic syndrome and 1.37 (1.03–1.82; P = 0.021) for type 2 diabetes. In conclusion, circulating miR-122 is strongly associated with the risk of developing metabolic syndrome and type 2 diabetes in the general population.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the joints. Untreated RA leads to a destruction of joints through the erosion of cartilage and bone. The loss of physical function is the consequence. Early treatment is important to control disease activity and to prevent joint destruction. Nowadays, different classes of drugs with different modes of action are available to control the inflammation and to achieve remission. In this review, we want to discuss differences and similarities of these different drugs.

MicroRNAs are small non-coding RNAs that are detectable in plasma and serum. Circulating levels of microRNAs have been measured in various studies related to cardiovascular disease. Heparin is a potential confounder of microRNA measurements due to its known interference with polymerase chain reactions. In this study, platelet-poor plasma was obtained from patients undergoing cardiac catheterisation for diagnostic coronary angiography, or for percutaneous coronary intervention, both before and after heparin administration. Heparin had pronounced effects on the assessment of the exogenous C. elegans spike-in control (decrease by approx. 3 cycles), which disappeared 6 hours after the heparin bolus. Measurements of endogenous microRNAs were less sensitive to heparin medication. Normalisation of individual microRNAs with the average cycle threshold value of all microRNAs provided a suitable alternative to normalisation with exogenous C. elegans spike-in control in this setting. Thus, both the timing of blood sampling relative to heparin dosing and the normalisation procedure are critical for reliable microRNA measurements in patients receiving intravenous heparin. This has to be taken into account when designing studies to investigate the relation of circulating microRNAs to acute cardiovascular events or coronary intervention.

MicroRNA (miRNA, miR) measurements in patients with coronary heart disease are hampered by the confounding effects of medication commonly used in cardiovascular patients such as statins, antiplatelet drugs, and heparin administration. Statins reduce the circulating levels of liver-derived miR-122. Antiplatelet medication attenuates the release of platelet-derived miRNAs. Heparin inhibits the polymerase chain reactions, in particular the amplification of the exogenous Caenorhabditis elegans spike-in control, thereby resulting in an artefactual rise of endogenous miRNAs. As these limitations have not been previously recognised, a reevaluation of the current miRNA literature, in particular of case–control studies in patients with cardiovascular disease or coronary interventions, is required.

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