Editor's Note: This article is in response to Zampetaki A, Willeit P, Tilling L et al. Prospective study on circulating microRNAs and risk of myocardial infarction. J Am Coll Cardiol 2012;60:290-299.

Article Summary

Background: Circulating miRNAs are emerging as potential biomarkers. We have previously identified a miRNA signature for type 2 diabetes in the general population.

Methods: We sought to explore the association between baseline levels of microRNAs (miRNAs) (1995) and incident myocardial infarction (1995-2005) in the Bruneck cohort and determine their cellular origin. A total of nineteen candidate miRNAs were quantified by real time polymerase chain reactions in 820 participants.

Results: In multivariable Cox regression analysis, three miRNAs were consistently and significantly related with incident myocardial infarction: miR-126 showed a positive association (multivariable hazard ratio 2.69 [95%CI 1.45-5.01], P=0.002) while miR-223 and miR-197 were inversely associated with disease risk (multivariable hazard ratio 0.47 [95% CI 0.29-0.75], P=0.002, and 0.56 [95% CI 0.32-0.96], P=0.036). When adding miRNAs to a base model considering the Framingham Risk Score for hard coronary heart disease endpoints, the gain in the C-index for miRNAs was higher than that for loge-transformed high-sensitivity CRP level (Δ 0.037 vs. Δ 0.005) and the integrated discrimination improvement, which does not rely on arbitrarily chosen cut-offs, was significant for cases, for controls as well as for the entire group. To determine their cellular origin, healthy volunteers were subject to limb ischaemia-reperfusion generated by thigh cuff inflation and plasma miRNA changes were analysed at baseline, at 10 min, 1h, 5h, 2 days and 7 days. Computational analysis using the temporal clustering by affinity propagation algorithm identified six distinct miRNA clusters. One cluster included all miRNAs associated with risk of future myocardial infarction. It was characterized by early (1h) and sustained activation (7 days) post ischaemia-reperfusion injury and consisted of miRNAs predominantly expressed in platelets.

Conclusions: In subjects with subsequent myocardial infarction differential co-expression patterns of circulating miRNAs occur around endothelial-enriched miR-126 with platelets being a major contributor to this miRNA signature.

Commentary/Perspective: Despite extensive studies and development of several risk prediction models, traditional risk factors fail to predict cardio- and cerebrovascular events in a large group of cases (25-50%). There are currently no good soluble biomarkers that could be used to identify patients who are at risk of developing acute manifestations of cardiovascular disease. Inflammatory markers such as high sensitivity CRP are used but lack specificity for the vasculature. Advanced imaging techniques are expensive and not suitable for population-wide screening. Thus, there is a clinical need for biomarkers that could complement the assessment of traditional risk factors.

Pioneering studies by Mitchell et al¹ have revealed the presence of endogenous microRNAs (miRNAs) in the circulation that are not cell-associated. Unlike messenger RNAs, miRNAs are stable in blood. They are protected from RNAse activity by microvesicles (exosomes, microparticles and apoptotic bodies), RNA-binding proteins (e.g. Ago2 complexes²) or lipoproteins (LDL and HDL³). Thus, besides their classical role as a delivery vehicle for cholesterol, lipoproteins may also act as a carrier or depot for endogenous miRNAs and facilitate their transport and delivery to recipient cells³. The very existence of a miRNA pool within the circulation is an exciting new aspect of current biology and is attracting considerable attention.

Our aim was to identify changes in circulating miRNAs that might precede subsequent cardiovascular events. As pointed out in the Editorial⁴ accompanying this publication⁵, the demonstration that a miRNA-based biomarker signature adds information to an established standard, the Framingham Risk Score for Hard Coronary Heart Disease, is one of the principal merits of this work suggesting that miRNAs may refine risk prediction for cardiovascular events. Also, miRNAs might offer certain advantages over other biomarkers: 1) Unlike messenger RNAs, miRNAs are stable in blood. 2) As nucleic acids, miRNAs can be both amplified and detected with high sensitivity and specificity⁶. Unlike protein-based biomarkers that tend to be measured individually, real-time PCR methodology allows the quantification of many miRNAs in a single experiment. 3) Because most circulating miRNAs are highly correlated, global patterns of expression should be studied by representing miRNA data as co-expression networks. Apart from their relative levels, it is the interaction / connectivity of a miRNA within the miRNA network that defines disease specific signatures⁷.

The cellular origin and the biological function of circulating miRNAs, however, are less clear⁸. Based on expression profiles after limb ischaemia-reperfusion injury, miR-126, miR-197 and miR-223 were part of one cluster that also included miR-21 and miR-24. All these miRNAs are highly expressed in platelets and platelet microparticles. This is, to our knowledge, the first time that the contribution of a specific cell type to circulating miRNAs has been defined by a controlled intervention. The present findings extend our previous observations in patients with diabetes⁹ and raise the possibility that the observed loss of several miRNAs, including miR-126, miR-197, miR-223, miR-24 and miR-21, may reflect abnormal platelet function in diabetic patients. It is noteworthy that several of the most abundant platelet miRNAs have previously been implicated in cardiovascular pathologies: miR-126 as master regulator of endothelial homeostasis and vascular integrity¹⁰, miR-21 as mediator of cardiac fibrosis¹¹ – although this is contested by others¹² – and miR-24 as inducer of endothelial apoptosis after myocardial infarction¹³. Although the functional importance of platelet miRNAs is not yet well defined¹⁴, it is conceivable that systemic inhibition of miRNAs, which are also abundant in platelets, may alter platelet function¹⁵ and contribute to the observed cardiovascular phenotypes. The presence of these miRNAs within platelets should be taken into account in future studies. Also, care must be taken in the design of case control studies for biomarker analysis¹⁶. Comparisons of circulating miRNAs between patients with manifest disease and healthy controls, for example, are likely to be confounded by medication, in particular anti-platelet therapy⁷.

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Keywords: Cardiovascular Diseases, MicroRNAs, Myocardial Infarction, Real-Time Polymerase Chain Reaction

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