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.

Prostacyclin is an antithrombotic hormone produced by the endothelium, whose production is dependent on cyclooxygenase (COX) enzymes of which two isoforms exist. It is widely believed that COX-2 drives prostacyclin production and that this explains the cardiovascular toxicity associated with COX-2 inhibition, yet the evidence for this relies on indirect evidence from urinary metabolites. Here we have used a range of experimental approaches to explore which isoform drives the production of prostacyclin in vitro and in vivo. Our data show unequivocally that under physiological conditions it is COX-1 and not COX-2 that drives prostacyclin production in the cardiovascular system, and that urinary metabolites do not reflect prostacyclin production in the systemic circulation. With the idea that COX-2 in endothelium drives prostacyclin production in healthy individuals removed, we must seek new answers to why COX-2 inhibitors increase the risk of cardiovascular events to move forward with drug discovery and to enable more informed prescribing advice.

There is considerable evidence that the potent vasoconstrictor endothelin-1 (ET-1) contributes to the pathogenesis of a variety of cardiovascular diseases. As such, pharmacological manipulation of the ET system might represent a promising therapeutic goal. Many clinical trials have assessed the potential of ET receptor antagonists in cardiovascular disease, the most positive of which have resulted in the licensing of the mixed ET receptor antagonist bosentan, and the selective ET A receptor antagonists, sitaxsentan and ambrisentan, for the treatment of pulmonary arterial hypertension (PAH). In contrast, despite encouraging data from in vitro and animal studies, outcomes in human heart failure have been disappointing, perhaps illustrating the risk of extrapolating preclinical work to man. Many further potential applications of these compounds, including resistant hypertension, chronic kidney disease, connective tissue disease and sub-arachnoid haemorrhage are currently being investigated in the clinic. Furthermore, experience from previous studies should enable improved trial design and scope remains for development of improved compounds and alternative therapeutic strategies. Although ET-converting enzyme inhibitors may represent one such alternative, there have been relatively few suitable compounds developed, and consequently, clinical experience with these agents remains extremely limited. Recent advances, together with an increased understanding of the biology of the ET system provided by improved experimental tools (including cell-specific transgenic deletion of ET receptors), should allow further targeting of clinical trials to diseases in which ET is involved and allow the therapeutic potential for targeting the ET system in cardiovascular disease to be fully realized.

Eicosanoids represent a diverse family of lipid mediators with fundamental roles in physiology and disease. Within the eicosanoid superfamily are prostanoids, which are specifically derived from arachidonic acid by the enzyme cyclooxygenase (COX). COX has two isoforms; COX-1 and COX-2. COX-2 is the therapeutic target for the nonsteroidal anti-inflammatory drug (NSAID) class of pain medications. Of the prostanoids, prostacyclin, first discovered by Sir John Vane in 1976, remains amongst the best studied and retains an impressive pedigree as one of the fundamental cardiovascular protective pathways. Since this time, we have learnt much about how eicosanoids, COX enzymes and prostacyclin function in the cardiovascular system, knowledge that has allowed us, for example, to harness the power of prostacyclin as therapy to treat pulmonary arterial hypertension and peripheral vascular disease. However, there remain many unanswered questions in our basic understanding of the pathways, and how they can be used to improve human health. Perhaps, the most important and controversial outstanding question in the field remains; 'how do NSAIDs produce their much publicized cardiovascular side-effects?' This review summarizes the history, biology and cardiovascular function of key eicosanoids with particular focus on prostacyclin and other COX products and discusses how our knowledge of these pathways can applied in future drug discovery and be used to explain the cardiovascular side-effects of NSAIDs.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system. cyclooxygenase | nonsteroidal antiinflammatory drugs | prostacyclin | cardiovascular | Vioxx C yclooxygenase (COX) converts arachidonic acid to prostanoids, which include prostaglandins (PGs), prostacyclin, and thromboxane. Prostanoids are important mediators that regulate diverse functions in the cardiovascular, gastrointestinal, urogenital, and nervous systems, as well as playing critical roles in immunity, inflammation and resolution of inflammation. There are two COX

P ulmonary arterial hypertension (PAH) is a rare but devastating disease, which is defined as a mean pulmonary artery pressure of ≥25 mm Hg with a normal pulmonary capillary wedge pressure. It is characterized by remodeling of the muscular, precapillary vessels, leading to an increase in pulmonary vascular resistance. The associated strain exerted on the right heart ultimately results in right heart failure and premature death. 1,2 Autoimmunity has long been implicated in PAH 3 and, most recently, evidence has emerged implicating interferon (IFN). 4 IFN is central to the innate immune response to viral infection, and 3 types have been identified; type I IFN (IFNα and IFNβ) that signals through a heterodimeric receptor consisting of IFNAR1 and IFNAR2, type II IFN (IFNγ) that signals through IFNGR1 and IFNGR2, and type III IFN (IFNλ) the receptor for which comprises interleukin (IL)10RB and IL28RA. In This Issue, see p 587Clinical Track © 2013 American Heart Association, Inc. Rationale: Evidence is increasing of a link between interferon (IFN) and pulmonary arterial hypertension (PAH).Conditions with chronically elevated endogenous IFNs such as systemic sclerosis are strongly associated with PAH. Furthermore, therapeutic use of type I IFN is associated with PAH. This was recognized at the 2013 World Symposium on Pulmonary Hypertension where the urgent need for research into this was highlighted.Objective: To explore the role of type I IFN in PAH. Methods and Results: Cells were cultured using standard approaches. Cytokines were measured by ELISA.Gene and protein expression were measured using reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemistry. The role of type I IFN in PAH in vivo was determined using type I IFN receptor knockout (IFNAR1 −/− ) mice. Human lung cells responded to types I and II but not III IFN correlating with relevant receptor expression. Type I, II, and III IFN levels were elevated in serum of patients with systemic sclerosis associated PAH. Serum interferon γ inducible protein 10 (IP10; CXCL10) and endothelin 1 were raised and strongly correlated together. IP10 correlated positively with pulmonary hemodynamics and serum brain natriuretic peptide and negatively with 6-minute walk test and cardiac index. Endothelial cells grown out of the blood of PAH patients were more sensitive to the effects of type I IFN than cells from healthy donors. PAH lung demonstrated increased IFNAR1 protein levels. IFNAR1 −/− mice were protected from the effects of hypoxia on the right heart, vascular remodeling, and raised serum endothelin 1 levels. Conclusions: These data indicate that type I IFN, via an action of IFNAR1, mediates PAH. (Circ Res. 2014;114:677-688.)Key Words: chemokine CXCL10 ■ endothelin-1 ■ IFNAR1 subunit, interferon alpha-beta receptor ■ inflammation ■ interferon type I ■ pulmonary arterial hypertension ■ scleroderma, systemic Original received July 18, 2013; revision received December 11, 2013; accepted December 13, 2013. In November 2013, the averag...

In the presence of strong P2Y 12 receptor blockade, aspirin provides little additional inhibition of platelet aggregation. J Thromb Haemost 2011; 9: 552-61.Summary. Background: Aspirin and antagonists of platelet ADP P2Y 12 receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y 12 receptors can inhibit thromboxane A 2 (TXA 2 )-dependent pathways of platelet activation independently of aspirin. Objectives: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y 12 receptor blockade. Methods: With the use of platelet-rich plasma from healthy volunteers, determinations were made in 96-well plates of platelet aggregation, TXA 2 production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP-6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1-10 lmol L ) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA 2 and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. Conclusions: P2Y 12 receptors are critical to the generation of irreversible aggregation through the TXA 2 -dependent pathway. As a result, strong P2Y 12 receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.

There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.