Antiplatelet drugs do not protect from platelet-leukocyte aggregation in coronary artery disease.

BACKGROUND: Despite advances in cardiovascular medicine, coronary artery disease (CAD) remains a leading cause of mortality. Among the pathophysiological features of this condition, platelet-leukocyte aggregates (PLAs) require further attention, either as diagnostic/prognostic disease markers or as potential interventional targets. OBJECTIVES: In this study, we characterized PLAs in patients with CAD. Primarily, we investigated the association of PLA levels with CAD diagnosis. In addition, the basal levels of platelet activation and degranulation were assessed in patients with CAD and controls, and their correlation with PLA levels was analyzed. Finally, the effect of antiplatelet treatments on circulating PLA numbers, basal platelet activation, and degranulation was studied in patients with CAD. METHODS: Participants were recruited at the Department of Cardiology of the University Heart and Vascular Centre Hamburg Eppendorf. Among patients admitted with severe chest pain, the diagnosis of CAD was made angiographically, and patients without CAD were used as controls. PLAs, platelet activation, and platelet degranulation were assessed by flow cytometry. RESULTS: Circulating PLAs and basal platelet degranulation levels were significantly higher in patients with CAD than in controls. Surprisingly, there was no significant correlation between PLA levels and platelet degranulation (or any other measured parameter). In addition, patients with CAD on antiplatelet therapy did not display lower PLA or platelet degranulation levels compared with those in controls. CONCLUSION: Overall, these data suggest a mechanism of PLA formation that is independent of platelet activation or degranulation and highlights the inefficiency of current antiplatelet treatments for the prevention of basal platelet degranulation and PLA formation.

The phosphodiesterase 2A controls lymphatic junctional maturation via cGMP-dependent notch signaling.

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.

Serial measurements of protein and microRNA biomarkers to specify myocardial infarction subtypes.

Background: While cardiac-specific troponin (cTn) allows for rapid diagnosis of acute type 1 myocardial infarction (T1MI), its performance to differentiate acute myocardial injury (AI) or type 2 myocardial infarction (T2MI) is limited. The objective was to combine biomarkers to improve discrimination of different myocardial infarction (MI) aetiologies. Methods: We determined levels of cardiac troponin T and I (cTnT, cTnI), cardiac myosin-binding protein C (cMyBP-C), NT-proBNP and ten miRNAs, known to be associated with cardiac pathology in a total of n = 495 serial plasma samples at three time points (on admission, after 1 h and 3 h) from 57 NSTEMI (non-ST-elevation myocardial infarction), 18 AI, and 31 STEMI patients, as defined by fourth universal definition of MI (UDMI4) and 59 control individuals. We then applied linear mixed effects model to compare the kinetics of all molecules in these MI sub-types. Results: Established (cTnT, cTnI) and novel (cMyBP-C) cardiac necrosis markers failed in differentiating T1MI vs T2MI at early time points. All cardiac necrosis markers were higher in T1MI than in T2MI at 3 h after admission. Muscle-enriched miRNAs (miR-1 and miR-133a) were correlated with cardiac necrosis protein markers and did not offer better discrimination. Established cardiac strain marker NT-proBNP differentiated AI and T1MI at all time points but failed to discriminate T2MI from T1MI. However, the combination of NT-proBNP and cTnT along with age returned an overall AUC of 0.76 [95 % CI 0.67-0.84] for differentiating T1MI, T2MI and AI. Conclusions: Rather than using single biomarkers of myocardial necrosis, a combination of clinical biomarkers for cardiac necrosis (troponin) and cardiac strain (NT-proBNP) might aid in differentiating T1MI, T2MI and AI.

The Tacrolimus Metabolism Rate and Dyslipidemia after Kidney Transplantation.

Fast tacrolimus (Tac) metabolism is associated with reduced survival rates after renal transplantation (RTx), mainly due to cardiovascular events. Because dyslipidemia is a leading cause of cardiovascular death, we hypothesized that most RTx patients do not achieve recommended target low-density lipoprotein cholesterol (LDL-C) levels (European cardiology society guidelines) and that fast Tac metabolizers have higher dyslipidemia rates. This study included RTx recipients who received initial immunosuppression with immediate-release tacrolimus (IR-Tac), mycophenolate, and prednisolone. Patients were grouped according to their Tac concentration-to-dose ratio (C/D ratio) 3 months after RTx. Dyslipidemia parameters were analyzed at RTx, 3 months, and 12 months after RTx. Statin use and renal function were documented in a 12-month follow-up, and death was documented in a 60-month follow-up. Ninety-six RTx recipients were divided into two groups: 31 fast Tac metabolizers (C/D ratio < 1.05 ng/mL·1/mg) and 65 slow metabolizers (C/D ratio ≥ 1.05 ng/mL·1/mg). There were no differences in triglyceride or cholesterol levels between groups at RTx, 3, and 12 months after RTx. A total of 93.5% of fast and 95.4% of slow metabolizers did not achieve target LDL-C levels (p = 0.657). Fast metabolizers developed lower renal function compared to slow metabolizers 12 months after RTx (p = 0.009). Fast metabolizers showed a 60 month survival rate of 96.8% compared to 94.7% in the slow metabolizer group (p = 0.811). As most RTx recipients do not reach recommended target LDL-C levels, individualized nutritional counseling and lipid-lowering therapy must be intensified. Fast Tac metabolism is associated with lower renal function after RTx, but does not play a significant role in dyslipidemia.

Biomarkers for Heart Failure Prognosis: Proteins, Genetic Scores and Non-coding RNAs.

Heart failure (HF) is a complex disease in which cardiomyocyte injury leads to a cascade of inflammatory and fibrosis pathway activation, thereby causing decrease in cardiac function. As a result, several biomolecules are released which can be identified easily in circulating body fluids. The complex biological processes involved in the development and worsening of HF require an early treatment strategy to stop deterioration of cardiac function. Circulating biomarkers provide not only an ideal platform to detect subclinical changes, their clinical application also offers the opportunity to monitor disease treatment. Many of these biomarkers can be quantified with high sensitivity; allowing their clinical application to be evaluated beyond diagnostic purposes as potential tools for HF prognosis. Though the field of biomarkers is dominated by protein molecules, non-coding RNAs (microRNAs, long non-coding RNAs, and circular RNAs) are novel and promising biomarker candidates that encompass several ideal characteristics required in the biomarker field. The application of genetic biomarkers as genetic risk scores in disease prognosis, albeit in its infancy, holds promise to improve disease risk estimation. Despite the multitude of biomarkers that have been available and identified, the majority of novel biomarker candidates are not cardiac-specific, and instead may simply be a readout of systemic inflammation or other pathological processes. Thus, the true value of novel biomarker candidates in HF prognostication remains unclear. In this article, we discuss the current state of application of protein, genetic as well as non-coding RNA biomarkers in HF risk prognosis.

Noncoding RNAs versus Protein Biomarkers in Cardiovascular Disease.

The development of more sensitive protein biomarker assays results in continuous improvements in detectability, extending the range of clinical applications to the detection of subclinical cardiovascular disease (CVD). However, these efforts have not yet led to improvements in risk assessment compared with existing risk scores. Noncoding RNAs (ncRNAs) have been assessed as biomarkers, and miRNAs have attracted most attention. More recently, other ncRNA classes have been identified, including long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs). Here, we compare emerging ncRNA biomarkers in the cardiovascular field with protein biomarkers for their potential in clinical application, focusing on myocardial injury.

Aspirin, clopidogrel and prasugrel monotherapy in patients with type 2 diabetes mellitus: a double-blind randomised controlled trial of the effects on thrombotic markers and microRNA levels.

BACKGROUND: Despite increased atherothrombotic risk in type 2 diabetes mellitus, (T2DM) the best preventative antithrombotic strategy remains undetermined. We defined the effects of three antiplatelet agents on functional readout and biomarker kinetics in platelet activation and coagulation in patients with T2DM. MATERIALS AND METHODS: 56 patients with T2DM were randomised to antiplatelet monotherapy with aspirin 75 mg once daily (OD), clopidogrel 75 mg OD or prasugrel 10 mg OD during three periods of a crossover study. Platelet aggregation (PA) was determined by light-transmittance aggregometry and P-selectin expression by flow cytometry. Markers of fibrin clot dynamics, inflammation and coagulation were measured. Plasma levels of 14 miRNA were assessed by quantitative polymerase chain reactions. RESULTS: Of the 56 patients, 24 (43%) were receiving aspirin for primary prevention of ischaemic events and 32 (57%) for secondary prevention. Prasugrel was the strongest inhibitor of ADP-induced PA (mean ± SD maximum response to 20µmol/L ADP 77.6 ± 8.4% [aspirin] vs. 57.7 ± 17.6% [clopidogrel] vs. 34.1 ± 14.1% [prasugrel], p < 0.001), P-selectin expression (30 µmol/L ADP; 45.1 ± 21.4% vs. 27.1 ± 19.0% vs. 14.1 ± 14.9%, p < 0.001) and collagen-induced PA (2 µg/mL; 62.1 ± 19.4% vs. 72.3 ± 18.2% vs. 60.2 ± 18.5%, p < 0.001). Fibrin clot dynamics and levels of coagulation and inflammatory proteins were similar. Lower levels of miR-24 (p = 0.004), miR-191 (p = 0.019), miR-197 (p = 0.009) and miR-223 (p = 0.014) were demonstrated during prasugrel-therapy vs. aspirin. Circulating miR-197 was lower in those cardiovascular disease during therapy with aspirin (p = 0.039) or prasugrel (p = 0.0083). CONCLUSIONS: Prasugrel monotherapy in T2DM provided potent platelet inhibition and reduced levels of a number of platelet-associated miRNAs. miR-197 is a potential marker of cardiovascular disease in this population. Clinical outcome studies investigating prasugrel monotherapy are warranted in individuals with T2DM. Trial registration EudraCT, 2009-011907-22. Registered 15 March 2010, https://www.clinicaltrialsregister.eu/ctr-search/trial/2009-011907-22/GB.

Biomarkers in primary prevention : Meaningful diagnosis based on biomarker scores?

Cardiovascular (CV) risk assessment is based on the utilization of risk scores, enabling clinicians to estimate an individual's risk to develop CV pathologies and events. Such risk scores comprise classic CV risk factors such as smoking, diabetes, hypertension, and blood cholesterol levels. Recently, other CV biomarkers such as cardiac troponins have been suggested and evaluated as alternative biomarkers not only in the acute diagnostic setting of myocardial infarction, but also as markers for risk stratification in the general population. In this review, we summarize the current knowledge on biomarkers in the field of primary prevention in cardiovascular disease (CVD). Furthermore, we present potential alternative biomarker-based strategies for CV risk assessment. In this respect we provide an outlook on the potential use of genomic variation as well as circulating non-coding RNAs to complement current risk assessment strategies so as to further personalize risk stratification in CVD.

Comparative Analysis of Circulating Noncoding RNAs Versus Protein Biomarkers in the Detection of Myocardial Injury.

RATIONALE: Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), circular RNAs (circRNAs), and long noncoding RNAs (lncRNAs), are proposed novel biomarkers of myocardial injury. Their release kinetics have not been explored without confounding by heparin nor has their relationship to myocardial protein biomarkers. OBJECTIVE: To compare ncRNA types in heparinase-treated samples with established and emerging protein biomarkers for myocardial injury. METHODS AND RESULTS: Screening of 158 circRNAs and 21 lncRNAs in human cardiac tissue identified 12 circRNAs and 11 lncRNAs as potential biomarkers with cardiac origin. Eleven miRNAs were included. At low spike-in concentrations of myocardial tissue, significantly higher regression coefficients were observed across ncRNA types compared with cardiac troponins and cMyBP-C (cardiac myosin-binding protein C). Heparinase treatment of serial plasma and serum samples of patients undergoing transcoronary ablation of septal hypertrophy removed spurious correlations between miRNAs in non-heparinase-treated samples. After transcoronary ablation of septal hypertrophy, muscle-enriched miRNAs (miR-1 and miR-133a) showed a steeper and earlier increase than cardiac-enriched miRNAs (miR-499 and miR-208b). Putative cardiac lncRNAs, including LIPCAR (long intergenic noncoding RNA predicting cardiac remodeling and survival), did not rise, refuting a predominant cardiac origin. Cardiac circRNAs remained largely undetectable. In a validation cohort of acute myocardial infarction, receiver operating characteristic curve analysis revealed noninferiority of cardiac-enriched miRNAs, but miRNAs failed to identify cases presenting with low troponin values. cMyBP-C was validated as a biomarker with highly sensitive properties, and the combination of muscle-enriched miRNAs with high-sensitive cardiac troponin T and cMyBP-C returned the highest area under the curve values. CONCLUSIONS: In a comparative assessment of ncRNAs and protein biomarkers for myocardial injury, cMyBP-C showed properties as the most sensitive cardiac biomarker while miRNAs emerged as promising candidates to integrate ncRNAs with protein biomarkers. Sensitivity of current miRNA detection is inferior to cardiac proteins but a multibiomarker combination of muscle-enriched miRNAs with cMyBP-C and cardiac troponins could open a new path of integrating complementary characteristics of different biomarker types.

STAT1 regulates macrophage number and phenotype and prevents renal fibrosis after ischemia-reperfusion injury.

Renal ischemia-reperfusion injury (IRI) leads to acute kidney injury or delayed allograft function, which predisposes to fibrosis in the native kidney or kidney transplant. Here we investigated the role of the signal transducer and activator of transcription 1 (STAT1) in inflammatory responses following renal IRI. Our study showed that a subsequent stimulation of Janus-activated kinase 2/STAT1 and Toll-like receptor 4 pathways led to greater STAT1 activation followed by increased cytokine transcription compared with single-pathway stimulation in murine renal tubular cells. Moreover, we observed increased activation of STAT1 under hypoxic conditions. In vivo, STAT1-/- mice displayed less acute tubular necrosis and decreased macrophage infiltration 24 h after renal ischemia. However, investigation of the healing phase (30 days after IRI) revealed significantly more fibrosis in STAT1-/- than in wild-type kidneys. In addition, we demonstrated increased macrophage infiltration in STAT1-/- kidneys. Flow cytometry analysis revealed that STAT1 deficiency drives an alternatively activated macrophage phenotype, which is associated with downregulated cluster of differentiation 80 expression, decreased intracellular reactive oxygen species production, and enhanced ability for phagocytosis. Furthermore, we detected immunohistochemically enhanced STAT1 expression in human renal allograft biopsies with no interstitial fibrosis/tubular atrophy (IF/TA) compared with specimens with severe IF/TA without specific etiology. Thus, STAT1 activation drives macrophages toward an alternatively activated phenotype and enhances fibrogenesis indicating a potential STAT1-driven protective mechanism in tissue repair after ischemic injury.

Inhibition of profibrotic microRNA-21 affects platelets and their releasate.

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21-null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-ß1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-ß1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-ß1 secretion, was identified as a direct target of miR-21. miR-21-null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-ß1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.

Circulating microRNAs strongly predict cardiovascular death in patients with coronary artery disease-results from the large AtheroGene study.

INTRODUCTION: Stratification for subsequent coronary events among patients with coronary artery disease (CAD) is of considerable interest because of the potential to guide secondary preventive therapies. Recently, we identified eight microRNAs (miRNAs), which facilitated acute coronary syndrome (ACS) diagnosis. In this study, we aimed to evaluate their potential role as prognostic biomarkers for cardiovascular disease. METHODS: The serum concentrations of eight candidate miRNAs -miR-19a, miR-19b, miR-132, miR-140-3p, miR-142-5p, miR-150, miR-186, and miR-210 were measured in a cohort of 1112 patients with documented CAD-including 430 patients with ACS and 682 patients with stable angina pectoris. Cardiovascular death was the main outcome measure. RESULTS: During a median follow-up of 4.0 years, most miRNAs reliably predicted cardiovascular death in ACS patients. Cox regression analyses indicated that in particular miR-132 (HR 2.85 per 1 SD increase, P = 0.022), miR-140-3p (HR 2.88 per 1 SD increase, P = 0.022), and miR-210 (HR 3.10 per 1 SD increase, P = 0.039) were able to precisely predict cardiovascular death. Circulating miR-132, miR-140-3p, and miR-210 clearly improved various model performance measures, including C-statistics (AUC [area under the receiver-operating characteristic curve] for miR-132: 0.737; AUC for miR-140-3p: 0.756; AUC for miR-210: 0.754). CONCLUSIONS: This is the largest study so far evaluating the prognostic value of circulating miRNAs in cardiovascular disease. Our study shows that single miRNAs derived from peripheral blood predict mortality in secondary prevention settings, and thereby represent valuable biomarkers for risk estimation in CAD.

microRNAs in cardiovascular disease - clinical application.

microRNAs (miRNAs) are well-known, powerful regulators of gene expression, and their potential to serve as circulating biomarkers is widely accepted. In cardiovascular disease (CVD), numerous studies have suggested miRNAs as strong circulating biomarkers with high diagnostic as well as prognostic power. In coronary artery disease (CAD) and heart failure (HF), miRNAs have been suggested as reliable biomarkers matching up to established protein-based such as cardiac troponins (cT) or natriuretic peptides. Also, in other CVD entities, miRNAs were identified as surprisingly specific biomarkers - with great potential for clinical applicability, especially in those entities that lack specific protein-based biomarkers such as atrial fibrillation (AF) and acute pulmonary embolism (APE). In this regard, miRNA signatures, comprising a set of miRNAs, yield high sensitivity and specificity. Attempts to utilize miRNAs as therapeutic agents have led to promising results. In this article, we review the clinical applicability of circulating miRNAs in CVD. We are giving an overview of miRNAs as biomarkers in numerous CVD entities to depict the variety of their potential clinical deployment. We illustrate the function of miRNAs by means of single miRNA examples in CVD.

Concomitant implantation of Impella® on top of veno-arterial extracorporeal membrane oxygenation may improve survival of patients with cardiogenic shock.

AIMS: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) support stabilizes patients with cardiogenic shock. Despite improved oxygenation and peripheral circulation, LV unloading may be impeded due to the increased afterload, resulting in a failing static left ventricle and in high mortality. METHODS AND RESULTS: We describe for the first time a large series of patients treated with the combination of VA-ECMO and Impella® compared with patients with VA-ECMO only. We retrospectively collected data on patients from two tertiary critical care referral centres. We enrolled 157 patients treated with VA-ECMO from January 2013 to April 2015: 123 received VA-ECMO support and 34 had concomitant treatment with VA-ECMO and Impella. A propensity-matching analysis was performed in a 2:1 ratio, resulting in 42 patients undergoing VA-ECMO alone (control group) compared with 21 patients treated with VA-ECMO and Impella. Patients in the VA-ECMO and Impella group had a significantly lower hospital mortality (47% vs. 80%, P < 0.001) and a higher rate of successful bridging to either recovery or further therapy (68% vs. 28%, P < 0.001) compared with VA-ECMO patients. A higher need for continuous veno-venous haemofiltration (48% vs. 19%, P = 0.02) and increased haemolysis (76% vs. 33%, P = 0.004) were reported in the study group due to higher survival. There was no difference in major bleeding rates between the two groups (VA-ECMO and Impella 38% vs. VA-ECMO 29%, P = 0.6). CONCLUSIONS: Concomitant treatment with VA-ECMO and Impella may improve outcome in patients with cardiogenic shock compared with VA-ECMO only. Nevertheless, randomized studies are needed to validate these promising results further.

EMMPRIN expression is involved in the development of interstitial fibrosis and tubular atrophy in human kidney allografts.

BACKGROUND: Matrix metalloproteinases (MMP) are involved in the development of interstitial fibrosis and tubular atrophy (IF/TA) in renal disease. The synthesis of MMP is activated by the extracellular matrix metalloproteinases inducer protein (EMMPRIN). To analyze the role of EMMPRIN in IF/TA, we retrospectively detected EMMPRIN expression in specimens of human renal allografts with various levels of IF/TA. METHODS: Immunohistochemistry was performed to detect EMMPRIN expression. In a retrospective analysis, a total cohort of 50 specimens were divided according to BANFF-classification into four subgroups (0-3): no, mild (≤ 25%), moderate (26-50%), or severe (>50%) IF/TA. Among other parameters, renal function was analyzed and compared to EMMPRIN expression. RESULTS: In 24 of 38 biopsies, we detected positive EMMPRIN staining. All nephrectomy (n = 12) samples were negative for EMMPRIN. Positive staining in the biopsy samples was detectable on the basolateral side of tubular epithelial cells. EMMPRIN staining was negatively correlated with IF/TA (p < 0.001). We found significant differences between the mean EMMPRIN expression in IF/TA groups 0 and 3 (p = 0.021) and groups 1 and 3 (p = 0.004). Furthermore, we found significant correlations between EMMPRIN staining and renal function. CONCLUSION: Our data suggest that EMMPRIN is involved in the pathophysiology of IF/TA.

microRNA-based diagnostics and therapy in cardiovascular disease-Summing up the facts.

Circulating microRNAs (miRNAs) are discussed as potential disease-specific biomarkers in cardiovascular disease. Their diagnostic value has been examined in numerous studies and animal models with respect to coronary artery disease (CAD) and myocardial infarction (MI) and the prognostic abilities of circulating miRNAs in risk stratification of future disease have been evaluated. Various miRNAs are described to complement protein-based biomarkers or classical risk factors in the diagnosis of CAD or MI and even represent potential new biomarkers in the discrimination of unstable angina pectoris (UAP). Signatures consisting of sets of multiple miRNAs seem to improve the predictive power compared to single miRNAs. Furthermore, the emerging field of miRNA-based therapeutics has reached cardiovascular research. The first promising in vitro results are raising hope for future clinical application. However, methods and material used for RNA isolation, miRNA detection and normalization steps still lack ways of standardization and need to be considered carefully. This article reviews the current knowledge of miRNAs in cardiovascular disease focusing on CAD and MI and will provide an overview regarding the use of circulating miRNAs as biomarkers and potential therapeutic targets in the field of CAD.

miRNA-197 and miRNA-223 Predict Cardiovascular Death in a Cohort of Patients with Symptomatic Coronary Artery Disease.

BACKGROUND: Circulating microRNAs (miRNAs) have been described as potential diagnostic biomarkers in cardiovascular disease and in particular, coronary artery disease (CAD). Few studies were undertaken to perform analyses with regard to risk stratification of future cardiovascular events. miR-126, miR-197 and miR-223 are involved in endovascular inflammation and platelet activation and have been described as biomarkers in the diagnosis of CAD. They were identified in a prospective study in relation to future myocardial infarction. OBJECTIVES: The aim of our study was to further evaluate the prognostic value of these miRNAs in a large prospective cohort of patients with documented CAD. METHODS: Levels of miR-126, miR-197 and miR-223 were evaluated in serum samples of 873 CAD patients with respect to the endpoint cardiovascular death. miRNA quantification was performed using real time polymerase chain reaction (RT-qPCR). RESULTS: The median follow-up period was 4 years (IQR 2.78-5.04). The median age of all patients was 64 years (IQR 57-69) with 80.2% males. 38.9% of the patients presented with acute coronary syndrome (ACS), 61.1% were diagnosed with stable angina pectoris (SAP). Elevated levels of miRNA-197 and miRNA-223 reliably predicted future cardiovascular death in the overall group (miRNA-197: hazard ratio (HR) 1.77 per one standard deviation (SD) increase (95% confidence interval (CI) 1.20; 2.60), p = 0.004, C-index 0.78; miRNA-223: HR 2.23 per one SD increase (1.20; 4.14), p = 0.011, C-index 0.80). In ACS patients the prognostic power of both miRNAs was even higher (miRNA-197: HR 2.24 per one SD increase (1.25; 4.01), p = 0.006, C-index 0.89); miRA-223: HR 4.94 per one SD increase (1.42; 17.20), p = 0.012, C-index 0.89). CONCLUSION: Serum-derived circulating miRNA-197 and miRNA-223 were identified as predictors for cardiovascular death in a large patient cohort with CAD. These results reinforce the assumption that circulating miRNAs are promising biomarkers with prognostic value with respect to future cardiovascular events.