Temo Barwari

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.

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.

Over the last few years, several groups have evaluated the potential of microRNAs (miRNAs) as biomarkers for cardiometabolic disease. In this review, we discuss the emerging literature on the role of miRNAs and other small noncoding RNAs in platelets and in the circulation, and the potential use of miRNAs as biomarkers for platelet activation. Platelets are a major source of miRNAs, YRNAs, and circular RNAs. By harnessing multiomics approaches, we may gain valuable insights into their potential function. Because not all miRNAs are detectable in the circulation, we also created a gene ontology annotation for circulating miRNAs using the gene ontology term extracellular space as part of blood plasma. Finally, we share key insights for measuring circulating miRNAs. We propose ways to standardize miRNA measurements, in particular by using platelet-poor plasma to avoid confounding caused by residual platelets in plasma or by adding RNase inhibitors to serum to reduce degradation. This should enhance comparability of miRNA measurements across different cohorts. We provide recommendations for future miRNA biomarker studies, emphasizing the need for accurate interpretation within a biological and methodological context.

BACKGROUND: The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the "vulnerable plaque," necessitating an improved approach for predicting onset of symptoms. METHODS: We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. RESULTS: Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. CONCLUSION: The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. FUNDING: UK: British Heart Foundation (BHF); King's BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London in partnership with King's College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.