Dorothee Kaudewitz

Circulating MicroRNAs as Novel Biomarkers for Platelet Activation

Rationale: MicroRNA (miRNA) biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far. Objective: To analyze changes in plasma miRNAs in response to antiplatelet therapy. Methods and Results: Profiling for 377 miRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum. Platelet-rich plasma showed markedly higher levels of miRNAs than serum and platelet-poor plasma. Few abundant platelet miRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared with platelet-poor plasma. In contrast, antiplatelet therapy significantly reduced miRNA levels. Using custom-made quantitative real-time polymerase chain reaction plates, 92 miRNAs were assessed in a dose-escalation study in healthy volunteers at 4 different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan quantitative real-time polymerase chain reaction assays (n=9). Validation was performed in an independent cohort of patients with symptomatic atherosclerosis (n=33), who received low-dose aspirin at baseline. Plasma levels of platelet miRNAs, such as miR-223, miR-191, and others, that is, miR-126 and miR-150, decreased on further platelet inhibition. Conclusions: Our study demonstrated a substantial platelet contribution to the circulating miRNA pool and identified miRNAs responsive to antiplatelet therapy. It also highlights that antiplatelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma miRNAs to cardiovascular disease.

Impact of intravenous heparin on quantification of circulating microRNAs in patients with coronary artery disease

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.

Association of MicroRNAs and YRNAs With Platelet Function

RATIONALE Platelets shed microRNAs (miRNAs). Plasma miRNAs change on platelet inhibition. It is unclear whether plasma miRNA levels correlate with platelet function. OBJECTIVE To link small RNAs to platelet reactivity. METHODS AND RESULTS Next-generation sequencing of small RNAs in plasma revealed 2 peaks at 22 to 23 and 32 to 33 nucleotides corresponding to miRNAs and YRNAs, respectively. Among YRNAs, predominantly, fragments of RNY4 and RNY5 were detected. Plasma miRNAs and YRNAs were measured in 125 patients with a history of acute coronary syndrome who had undergone detailed assessment of platelet function 30 days after the acute event. Using quantitative real-time polymerase chain reactions, 92 miRNAs were assessed in patients with acute coronary syndrome on different antiplatelet therapies. Key platelet-related miRNAs and YRNAs were correlated with platelet function tests. MiR-223 (rp=0.28; n=121; P=0.002), miR-126 (rp=0.22; n=121; P=0.016), and other abundant platelet miRNAs and YRNAs showed significant positive correlations with the vasodilator-stimulated phosphoprotein phosphorylation assay. YRNAs, miR-126, and miR-223 were also among the small RNAs showing the greatest dependency on platelets and strongly correlated with plasma levels of P-selectin, platelet factor 4, and platelet basic protein in the population-based Bruneck study (n=669). A single-nucleotide polymorphism that facilitates processing of pri-miR-126 to mature miR-126 accounted for a rise in circulating platelet activation markers. Inhibition of miR-126 in mice reduced platelet aggregation. MiR-126 directly and indirectly affects ADAM9 and P2Y12 receptor expression. CONCLUSIONS Levels of platelet-related plasma miRNAs and YRNAs correlate with platelet function tests in patients with acute coronary syndrome and platelet activation markers in the general population. Alterations in miR-126 affect platelet reactivity.

Circulating MicroRNA-122 is associated with the risk of new-onset metabolic syndrome and type 2 diabetes

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.

MicroRNA Biomarkers for Coronary Artery Disease

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.

PLASMA MICRORNAS AS BIOMARKERS FOR PLATELET INHIBITION

Introduction MicroRNAs (miRNAs) are small non-coding RNAs that are also detectable in the blood. Circulating levels of miRNAs have been measured in various epidemiological studies, but the effects of medication are unclear. To address these uncertainties, we aimed to (1) compare the levels of miRNAs in different sample types, (2) assess how miRNAs change in response to platelet inhibition and (3) correlate their plasma levels with existing markers of platelet activation. Methods MiRNAs were assessed by real-time polymerase chain reaction in the following cohorts: Study population Mean age, y Male, % 9 volunteers participating in a dose-escalation study <40 100% 33 patients with symptomatic carotid stenosis 72.1 ± 1.9 82% 129 patients post myocardial infarction 59.7 ± 10.4 81% Result (1) Profiling for 377 miRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum. Platelet-rich plasma showed markedly higher levels of miRNAs than serum and platelet-poor plasma. Few abundant platelet miRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared with platelet-poor plasma. (2) Antiplatelet therapy significantly reduced miRNA levels. Using custom-made quantitative real-time polymerase chain reaction plates, 92 miRNAs were assessed in a dose-escalation study in healthy volunteers at 4 different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan quantitative real-time polymerase chain reaction assays. Validation was performed in an independent cohort of patients with symptomatic atherosclerosis, who received low-dose aspirin at baseline. Plasma levels of platelet miRNAs, such as miR-223, miR-191, and others, that is, miR-126 and miR-150, decreased on further platelet inhibition. (3) In a cohort of patients on dual anti-platelet therapy 30 days post myocardial infarction, plasma levels of most platelet miRNAs were positively correlated to the vasodilator-stimulated phosphoprotein phosphorylation (VASP) assay. Levels of miR-126 were also significantly associated with results from the VerifyNow P2Y12aggregation assay. No association was observed with optical aggregometry either with arachidonic acid or with ADP. Conclusions Our study provides evidence for a correlation of plasma miRNAs with commonly used methods in platelet testing. It highlights that miRNA levels are sensitive to medication, in particular antiplatelet therapy, and preparation of blood samples. This has to be taken into account when designing a study to investigate the relation of circulating miRNAs with cardiovascular disease.