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...

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.

Rationale The pathogenesis of PAH remains unclear. The four microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. Objective To elucidate the transcriptional regulation of the miR-143/145 cluster, and the role of miR-143 in PAH. Methods and Results We identified the promoter region that regulates miR-143/145 miRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signalling pathways, including estrogens receptor (ER), liver X factor/retinoic X receptor (LXR/RXR), TGF-β (Smads), and hypoxia (HRE) that regulated levels of all pri-miR stem loop transcription and resulting miRNA expression. We observed that miR-143-3p is selectively upregulated compared to miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMCs-derived exosomes. Using assays with pulmonary arterial endothelial cells (PAECs) we demonstrated a paracrine pro-migratory and pro-angiogenic effect of miR-143-3p enriched exosomes from PASMC. Quantitative PCR and in situ hybridisation showed elevated expression of miR-143 in calf models of PAH as well as in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role for miR-143 in experimental PH in vivo in miR-143−/− and antimiR143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings. Conclusions miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while inhibition of miR-143-3p blocked experimental PH. Taken together these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology.

Rationale: MicroRNAs (miRNAs), in particular miR-29b and miR-30c, have been implicated as important regulators of cardiac fibrosis. Objective: To perform a proteomics comparison of miRNA effects on extracellular matrix secretion by cardiac fibroblasts. Methods and Results: Mouse cardiac fibroblasts were transfected with pre-/anti-miR of miR-29b and miR-30c, and their conditioned medium was analyzed by mass spectrometry. miR-29b targeted a cadre of proteins involved in fibrosis, including multiple collagens, matrix metalloproteinases, and leukemia inhibitory factor, insulin-like growth factor 1, and pentraxin 3, 3 predicted targets of miR-29b. miR-29b also attenuated the cardiac fibroblast response to transforming growth factor-β. In contrast, miR-30c had little effect on extracellular matrix production but opposite effects regarding leukemia inhibitory factor and insulin-like growth factor 1. Both miRNAs indirectly affected cardiac myocytes. On transfection with pre–miR-29b, the conditioned medium of cardiac fibroblasts lost its ability to support adhesion of rat ventricular myocytes and led to a significant reduction of cardiac myocyte proteins (α-actinin, cardiac myosin-binding protein C, and cardiac troponin I). Similarly, cardiomyocytes derived from mouse embryonic stem cells atrophied under pre–miR-29 conditioned medium, whereas pre–miR-30c conditioned medium had a prohypertrophic effect. Levels of miR-29a, miR-29c, and miR-30c, but not miR-29b, were significantly reduced in a mouse model of pathological but not physiological hypertrophy. Treatment with antagomiRs to miR-29b induced excess fibrosis after aortic constriction without overt deterioration in cardiac function. Conclusions: Our proteomic analysis revealed novel molecular targets of miRNAs that are linked to a fibrogenic cardiac phenotype. Such comprehensive screening methods are essential to define the concerted actions of miRNAs in cardiovascular disease.

Rationale: Despite improved understanding of the underlying genetics, pulmonary arterial hypertension (PAH) remains a severe disease. Extensive remodeling of small pulmonary arteries, including proliferation of pulmonary artery smooth muscle cells (PASMCs), characterizes PAH. MicroRNAs (miRNAs) are noncoding RNAs that have been shown to play a role in vascular remodeling. Objective:We assessed the role of miR-145 in PAH. Methods and Results:We localized miR-145 in mouse lung to smooth muscle. Using quantitative PCR, we demonstrated increased expression of miR-145 in wild-type mice exposed to hypoxia. PAH was evaluated in miR-145 knockout and mice treated with anti-miRs via measurement of systolic right ventricular pressure, right ventricular hypertrophy, and percentage of remodeled pulmonary arteries. miR-145 deficiency and anti-miR-mediated reduction resulted in significant protection from the development of PAH. In contrast, miR-143 anti-miR had no effect. Furthermore, we observed upregulation of miR-145 in lung tissue of patients with idiopathic and heritable PAH compared with unaffected control subjects and demonstrated expression of miR-145 in SMC of remodeled vessels from such patients. Finally, we show elevated levels of miR-145 expression in primary PASMCs cultured from patients with BMPR2 mutations and also in the lungs of BMPR2-deficient mice. Key Words: pulmonary hypertension Ⅲ hypoxia Ⅲ molecular biology Ⅲ smooth muscle cells Ⅲ microRNA Ⅲ smooth muscle differentiation Ⅲ remodeling P ulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries (PAs), characterized by an increase in PA pressure and vascular remodeling leading to a progressive increase in pulmonary vascular resistance. 1 The consequence of vascular obliteration is right heart failure and high mortality. 2,3 Germline mutations in the gene coding for the bone morphogenetic protein (BMP) type-2 receptor (BMPR2), a receptor for the transforming growth factor (TGF)-␤ superfamily, have been identified in approximately 70% of patients with the heritable form of PAH (hPAH). 4 Moreover, BMPR2 expression is markedly reduced in PAH cases in the absence of mutations in this gene (idiopathic PAH, iPAH). In pulmonary artery smooth muscle cells (PASMCs), mutations in BMPR2 are associated with an abnormal growth response to BMPs and TGF-␤. 5 In pulmonary artery endothelial cells (PAECs), these mutations increase the susceptibility of cells to apoptosis. 4,6 The absence of BMPR2 mutations in some families and in the majority of iPAH cases suggests that further pathological mechanisms still need to be identified. One of the main histopathologic features common to all forms of PAH is the accumulation of cells expressing smooth muscle specific ␣-actin (SMA) in Original received February 23, 2012; revision received June 11, 2012; accepted June 11, 2012. In May 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.0 days. MicroRNAs (miRNAs) are a class of ...

Transforming growth factor (TGF)-

Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti-miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-b signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-b blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-b signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney.

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.