Time from blood draw to multiple electrode aggregometry and association with platelet reactivity.

Results from multiple electrode aggregometry (MEA) may vary according to pre-analytic factors. This study aimed to analyze the association of time from blood draw to MEA in patients undergoing percutaneous coronary intervention (PCI). In this observational single-center cohort study, platelet aggregation (aggregation units, U) was quantified by MEA (Multiplate Analyzer) after stimulation with adenosine diphosphate (ADP; final concentration [Fc] 6.4 µM), thrombin receptor activating peptide (TRAP; Fc 32 µM), or arachidonic acid (AA; Fc 0.5 mM) in patients treated with ASA and clopidogrel following PCI. High on-clopidogrel platelet reactivity (HPR) was defined as ADP-induced platelet aggregation ≥ 46 U. The manufacturer recommends performing the analysis within 30-180 min after blood draw. Patients were grouped according to the time from blood draw to MEA: 30-180 min, < 30 min, or > 180 min. Platelet function of 273 patients with coronary artery disease undergoing PCI with dual antiplatelet therapy was analyzed. The median age was 72 years (interquartile range, IQR 62-79) and 179 (66%) were male. Median ADP-, TRAP-, and AA-induced aggregation was 25 (IQR 18-36) U, 79 (IQR 63-96) U, and 12 (IQR 7-18) U, respectively. For those analyzed within 30-180 min from blood draw, no significant correlation of time from blood draw to MEA was observed 1) ADP (r = - 0.04, p = 0.51); 2) TRAP (r = - 0.06, p = 0.32); 3) AA (r = - 0.03, p = 0.67). In patients undergoing percutaneous coronary intervention and treated with dual antiplatelet therapy, the time from blood draw to multiple electrode aggregometry does not correlate with ADP- induced aggregation when the measurement occurred within the recommended time interval of 30-180 min after blood draw.

BMPER Improves Vascular Remodeling and the Contractile Vascular SMC Phenotype.

Dedifferentiated vascular smooth muscle cells (vSMCs) play an essential role in neointima formation, and we now aim to investigate the role of the bone morphogenetic protein (BMP) modulator BMPER (BMP endothelial cell precursor-derived regulator) in neointima formation. To assess BMPER expression in arterial restenosis, we used a mouse carotid ligation model with perivascular cuff placement. Overall BMPER expression after vessel injury was increased; however, expression in the tunica media was decreased compared to untreated control. Consistently, BMPER expression was decreased in proliferative, dedifferentiated vSMC in vitro. C57BL/6_Bmper+/- mice displayed increased neointima formation 21 days after carotid ligation and enhanced expression of Col3A1, MMP2, and MMP9. Silencing of BMPER increased the proliferation and migration capacity of primary vSMCs, as well as reduced contractibility and expression of contractile markers, whereas stimulation with recombinant BMPER protein had the opposite effect. Mechanistically, we showed that BMPER binds insulin-like growth factor-binding protein 4 (IGFBP4), resulting in the modulation of IGF signaling. Furthermore, perivascular application of recombinant BMPER protein prevented neointima formation and ECM deposition in C57BL/6N mice after carotid ligation. Our data demonstrate that BMPER stimulation causes a contractile vSMC phenotype and suggest that BMPER has the potential for a future therapeutic agent in occlusive cardiovascular diseases.

Cardiomyocyte-specific miR-100 overexpression preserves heart function under pressure overload in mice and diminishes fatty acid uptake as well as ROS production by direct suppression of Nox4 and CD36.

MicroRNAs are key regulators of the cardiac response to injury. MiR-100 has recently been suggested to be involved in different forms of heart failure, but functional studies are lacking. In the present study, we examined the impact of transgenic miR-100 overexpression on cardiac structure and function during physiological aging and pathological pressure-overload-induced heart failure in mice after transverse aortic constriction surgery. MiR-100 was moderately upregulated after induction of pressure overload in mice. While in our transgenic model the cardiomyocyte-specific overexpression of miR-100 did not result in an obvious cardiac phenotype in unchallenged mice, the transgenic mouse strain exhibited less left ventricular dilatation and a higher ejection fraction than wildtype animals, demonstrating an attenuation of maladaptive cardiac remodeling by miR-100. Cardiac transcriptome analysis identified a repression of several regulatory genes related to cardiac metabolism, lipid peroxidation, and production of reactive oxygen species (ROS) by miR-100 overexpression, possibly mediating the observed functional effects. While the modulation of ROS-production seemed to be indirectly affected by miR-100 via Alox5-and Nox4-downregulation, we demonstrated that miR-100 induced a direct repression of the scavenger protein CD36 in murine hearts resulting in a decreased uptake of long-chain fatty acids and an alteration of mitochondrial respiratory function with an enhanced glycolytic state. In summary, we identified miR-100 as a modulator of cardiac metabolism and ROS production without an apparent cardiac phenotype at baseline but a protective effect under conditions of pressure-overload-induced cardiac stress, providing new insight into the mechanisms of heart failure.

eif4ebp3l-A New Affector of Zebrafish Angiogenesis and Heart Regeneration?

The eukaryotic initiation factor 4E binding protein (4E-BP) family is involved in translational control of cell proliferation and pro-angiogenic factors. The zebrafish eukaryotic initiation factor 4E binding protein 3 like (eif4ebp3l) is a member of the 4E-BPs and responsible for activity-dependent myofibrillogenesis, but whether it affects cardiomyocyte (CM) proliferation or heart regeneration is unclear. We examined eif4ebp3l during zebrafish vascular development and heart regeneration post cryoinjury in adult zebrafish. Using morpholino injections we induced silencing of eif4ebp3l in zebrafish embryos, which led to increased angiogenesis at 94 h post fertilization (hpf). For investigation of eif4ebp3l in cardiac regeneration, zebrafish hearts were subjected to cryoinjury. Regenerating hearts were analyzed at different time points post-cryoinjury for expression of eif4ebp3l by in situ hybridization and showed strongly decreased eif4ebp3l expression in the injured area. We established a transgenic zebrafish strain, which overexpressed eif4ebp3l under the control of a heat-shock dependent promotor. Overexpression of eif4ebp3l during zebrafish heart regeneration caused only macroscopically a reduced amount of fibrin at the site of injury. Overall, these findings demonstrate that silencing of eif4ebp3l has pro-angiogenic properties in zebrafish vascular development and when eif4ebp3l is overexpressed, fibrin deposition tends to be altered in zebrafish cardiac regeneration after cryoinjury.

MiR-100 overexpression attenuates high fat diet induced weight gain, liver steatosis, hypertriglyceridemia and development of metabolic syndrome in mice.

BACKGROUND: Diet-induced obesity can result in the development of a diverse spectrum of cardiovascular and metabolic diseases, including type 2 diabetes, dyslipidemia, non-alcoholic liver steatosis and atherosclerotic disease. MicroRNAs have been described to be important regulators of metabolism and disease development. METHODS: In the current study, we investigated the effects of ubiquitous miR-100 overexpression on weight gain and the metabolic phenotype in a newly generated transgenic mouse strain under normal chow and high fat diet and used microarray expression analysis to identify new potential target genes of miR-100. RESULTS: While transgenic overexpression of miR-100 did not significantly affect weight and metabolism under a normal diet, miR-100 overexpressing mice showed a reduced weight gain under a high fat diet compared to wildtype mice, despite an equal calorie intake. This was accompanied by less visceral and subcutaneous fat development and lover serum LDL cholesterol. In addition, transgenic miR-100 mice were more glucose tolerant and insulin sensitive and demonstrated increased energy expenditure under high fat diet feeding. A comprehensive gene expression profiling revealed the differential expression of several genes involved in lipid storage- and metabolism, among them CD36 and Cyp4A14. Our data showed a direct regulation of CD36 by miR-100, leading to a reduced fatty acid uptake in primary hepatocytes overexpressing miR-100 and the downregulation of several downstream mediators of lipid metabolism such as ACC1, FABP4, FAS and PPARγ in the liver. CONCLUSIONS: Our findings demonstrate a protective role of miR-100 in high fat diet induced metabolic syndrome and liver steatosis, partially mediated by the direct repression of CD36 and attenuation of hepatic lipid storage, implicating miR-100 as a possible therapeutic target in liver steatosis.

A DARPin targeting activated Mac-1 is a novel diagnostic tool and potential anti-inflammatory agent in myocarditis, sepsis and myocardial infarction.

The monocyte ß2-integrin Mac-1 is crucial for leukocyte-endothelium interaction, rendering it an attractive therapeutic target for acute and chronic inflammation. Using phage display, a Designed-Ankyrin-Repeat-Protein (DARPin) was selected as a novel binding protein targeting and blocking the αM I-domain, an activation-specific epitope of Mac-1. This DARPin, named F7, specifically binds to activated Mac-1 on mouse and human monocytes as determined by flow cytometry. Homology modelling and docking studies defined distinct interaction sites which were verified by mutagenesis. Intravital microscopy showed reduced leukocyte-endothelium adhesion in mice treated with this DARPin. Using mouse models of sepsis, myocarditis and ischaemia/reperfusion injury, we demonstrate therapeutic anti-inflammatory effects. Finally, the activated Mac-1-specific DARPin is established as a tool to detect monocyte activation in patients receiving extra-corporeal membrane oxygenation, as well as suffering from sepsis and ST-elevation myocardial infarction. The activated Mac-1-specific DARPin F7 binds preferentially to activated monocytes, detects inflammation in critically ill patients, and inhibits monocyte and neutrophil function as an efficient new anti-inflammatory agent.

MicroRNA-100 Suppresses Chronic Vascular Inflammation by Stimulation of Endothelial Autophagy.

RATIONALE: The interaction of circulating cells within the vascular wall is a critical event in chronic inflammatory processes, such as atherosclerosis, but the control of the vascular inflammatory state is still largely unclear. OBJECTIVE: This study was undertaken to characterize the function of the endothelial-enriched microRNA miR-100 during vascular inflammation and atherogenesis. METHODS AND RESULTS: Based on a transcriptome analysis of endothelial cells after miR-100 overexpression, we identified miR-100 as a potent suppressor of endothelial adhesion molecule expression, resulting in attenuated leukocyte-endothelial interaction in vitro and in vivo as shown by flow cytometry and intravital imaging. Mechanistically, miR-100 directly repressed several components of mammalian target of rapamycin complex 1-signaling, including mammalian target of rapamycin and raptor, which resulted in a stimulation of endothelial autophagy and attenuated nuclear factor κB signaling in vitro and in vivo. In a low-density lipoprotein receptor-deficient atherosclerotic mouse model, pharmacological inhibition of miR-100 resulted in enhanced plaque lesion formation and a higher macrophage content of the plaque, whereas a systemic miR-100 replacement therapy had protective effects and attenuated atherogenesis, resulting in a decrease of plaque area by 45%. Finally, analysis of miR-100 expression in >70 samples obtained during carotid endarterectomy revealed that local miR-100 expression was inversely correlated with inflammatory cell content in patients. CONCLUSIONS: In summary, we describe an anti-inflammatory function of miR-100 in the vascular response to injury and inflammation and identify an important novel modulator of mammalian target of rapamycin signaling and autophagy in the vascular system. Our findings of miR-100 as a potential protective anti-athero-miR suggest that the therapeutic replacement of this microRNA could be a potential strategy for the treatment of chronic inflammatory diseases, such as atherosclerosis, in the future.

Cardiomyocyte microvesicles: proinflammatory mediators after myocardial ischemia?

Myocardial infarction is a frequent complication of cardiovascular disease leading to high morbidity and mortality worldwide. Elevated C-reactive protein (CRP) levels after myocardial infarction are associated with heart failure and poor prognosis. Cardiomyocyte microvesicles (CMV) are released during hypoxic conditions and can act as mediators of intercellular communication. MicroRNA (miRNA) are short non-coding RNA which can alter cellular mRNA-translation. Microvesicles (MV) have been shown to contain distinct patterns of miRNA from their parent cells which can affect protein expression in target cells. We hypothesized that miRNA containing CMV mediate hepatic CRP expression after cardiomyocyte hypoxia. H9c2-cells were cultured and murine cardiomyocytes were isolated from whole murine hearts. H9c2- and murine cardiomyocytes were exposed to hypoxic conditions using a hypoxia chamber. Microvesicles were isolated by differential centrifugation and analysed by flow cytometry. Next-generation-sequencing was performed to determine the miRNA-expression profile in H9c2 CMV compared to their parent cells. Microvesicles were incubated with a co-culture model of the liver consisting of THP-1 macrophages and HepG2 cells. IL-6 and CRP expression in the co-culture was assessed by qPCR and ELISA. CMV contain a distinct pattern of miRNA compared to their parent cells including many inflammation-related miRNA. CMV induced IL-6 expression in THP-1 macrophages alone and CRP expression in the hepatic co-culture model. MV from hypoxic cardiomyocytes can mediate CRP expression in a hepatic co-culture model. Further studies will have to show whether these effects are reproducible in-vivo.

Cardiac Endothelial Cell Transcriptome.

OBJECTIVE: Endothelial cells (ECs) are a highly specialized cell type with marked diversity between different organs or vascular beds. Cardiac ECs are an important player in cardiac physiology and pathophysiology but are not sufficiently characterized yet. Thus, the aim of the present study was to analyze the cardiac EC transcriptome. APPROACH AND RESULTS: We applied fluorescence-assisted cell sorting to isolate pure ECs from adult mouse hearts. RNAseq revealed 1288 genes predominantly expressed in cardiac ECs versus heart tissue including several transcription factors. We found an overrepresentation of corresponding transcription factor binding motifs within the promotor region of EC-enriched genes, suggesting that they control the EC transcriptome. Cardiac ECs exhibit a distinct gene expression profile when compared with renal, cerebral, or pulmonary ECs. For example, we found the Meox2/Tcf15, Fabp4, and Cd36 signaling cascade higher expressed in cardiac ECs which is a key regulator of fatty acid uptake and involved in the development of atherosclerosis. CONCLUSIONS: The results from this study provide a comprehensive resource of gene expression and transcriptional control in cardiac ECs. The cardiac EC transcriptome exhibits distinct differences in gene expression compared with other cardiac cell types and ECs from other organs. We identified new candidate genes that have not been investigated in ECs yet as promising targets for future evaluation.

Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells.

The serine protease high-temperature-required protein A2 (HtrA2) has been identified as a key intracellular molecule promoting apoptosis in cells during ischemia reperfusion (IR) injury. IR injury in ST-segment elevation myocardial infarction (STEMI) contributes to overall myocardial damage. HtrA2 has further been shown to be significantly increased in the serum of patients with STEMI. In the present pilot study, we use human umbilical vein endothelial cells (HUVECs) to investigate whether extracellular HtrA2 induces apoptosis using Annexin V staining. Furthermore, we examine whether HtrA2 is released extracellularly after staurosporine-induced apoptosis using ELISA. We find that HtrA2 is released upon induction of apoptosis by staurosporine into the cell culture medium. Furthermore, treatment of HUVECs with extracellular HtrA2-induces apoptosis, while the addition of anti-HtrA2 antibodies reduces both HtrA2- and staurosporine-induced endothelial cell apoptosis. In conclusion, we show here that extracellular HtrA2 induces apoptosis in human endothelial cells, although the exact molecular mechanisms have to be investigated in future.

Mineralocorticoids in the heart and vasculature: new insights for old hormones.

The mineralocorticoid aldosterone is a key regulator of water and electrolyte homeostasis. Numerous recent developments have advanced the field of mineralocorticoid pharmacology­namely, clinical trials have shown the beneficial effects of aldosterone antagonists in chronic heart failure and post-myocardial infarction treatment. Experimental studies using cell type-specific gene targeting of the mineralocorticoid receptor (MR) gene in mice have revealed the importance of extrarenal aldosterone signaling in cardiac myocytes, endothelial cells, vascular smooth cells, and macrophages. In addition, several molecular pathways involving signal transduction via the classical MR as well as the G protein-coupled receptor GPER mediate the diverse spectrum of effects of aldosterone on cells. This knowledge has initiated the development of new pharmacological ligands to specifically interfere with targets on different levels of aldosterone signaling. For example, aldosterone synthase inhibitors such as LCI699 and the novel nonsteroidal MR antagonist BAY 94-8862 have been tested in clinical trials. Interference with the interaction between MR and its coregulators seems to be a promising strategy toward the development of selective MR modulators.

The effect of oxygen in Sirt3-mediated myocardial protection: a proof-of-concept study in cultured cardiomyoblasts.

Sirtuin 3 is a nicotinamide adenine dinucleotide dependent mitochondrial deacetylase that governs mitochondrial metabolism and oxidative defense. The demise in myocardial function following myocardial ischemia has been associated with mitochondrial dysfunction. Sirt3 maintains myocardial contractile function and protects from cardiac hypertrophy. The role of Sirt3 in ischemia is controversial. Our objective was to understand, under what circumstances Sirt3 is protective in different facets of ischemia, using an in vitro proof-of-concept approach based on simulated ischemia in cultured cardiomyoblasts. Cultured H9c2 cardiomyoblasts were subjected to hypoxia and/or serum deprivation, the combination of which we refer to as simulated ischemia. Apoptosis, as assessed by Annexin V staining in life-cell imaging and propidium-iodide inclusion in flow cytometry, was enhanced following simulated ischemia. Interestingly, serum deprivation was a stronger trigger of apoptosis than hypoxia. Knockdown of Sirt3 further increased apoptosis upon serum deprivation, whereas no such effect occurred upon additional hypoxia. Similarly, only upon serum deprivation but not upon simulated ischemia, silencing of Sirt3 led to a deterioration of mitochondrial function in extracellular flux analysis. In the absence of oxygen these Sirt3-dependent effects were abolished. These data indicate, that Sirt3-mediated myocardial protection is oxygen-dependent. Thus, mitochondrial respiration takes center-stage in Sirt3-dependent prevention of stress-induced myocardial damage.

MicroRNA-155 Exerts Cell-Specific Antiangiogenic but Proarteriogenic Effects During Adaptive Neovascularization.

BACKGROUND: Adaptive neovascularization after arterial occlusion is an important compensatory mechanism in cardiovascular disease and includes both the remodeling of pre-existing vessels to collateral arteries (arteriogenesis) and angiogenic capillary growth. We now aimed to identify regulatory microRNAs involved in the modulation of neovascularization after femoral artery occlusion in mice. METHODS AND RESULTS: Using microRNA-transcriptome analysis, we identified miR-155 as a downregulated microRNA during hindlimb ischemia. Correspondingly, inhibition of miR-155 in endothelial cells had a stimulatory effect on proliferation and angiogenic tube formation via derepression of its direct target gene angiotensin II type 1 receptor. Surprisingly, miR-155-deficient mice showed an unexpected phenotype in vivo, with a strong reduction of blood flow recovery after femoral artery ligation (arteriogenesis) dependent on the attenuation of leukocyte-endothelial interaction and a reduction of proarteriogenic cytokine expression. Consistently, miR-155-deficient macrophages exhibit a specific alteration of the proarteriogenic cytokine expression profile, which is partly mediated by the direct miR-155 target gene SOCS-1. CONCLUSIONS: Our data demonstrate that miR-155 exerts an antiangiogenic but proarteriogenic function in the regulation of neovascularization via the suppression of divergent cell-specific target genes and that its expression in both endothelial and bone marrow-derived cells is essential for arteriogenesis in response to hindlimb ischemia in mice.

Aortic root volume is associated with contained rupture of the aortic annulus in balloon-expandable transcatheter aortic valve replacement.

BACKGROUND: Aortic annulus rupture is a rare, but potentially fatal complication of transcatheter aortic valve replacement (TAVR), especially when it occurs by balloon-expandable devices. In order to improve the predictability of procedures and avoid ruptures we investigated whether or not the aortic root volume measures is a useful indicator of risk, and if it could be useful for the prosthesis size selection. METHODS AND RESULTS: From a retrospective series of 172 TAVR patients, seven experienced contained aortic annulus ruptures. The receiver operating curves were used to illustrate sensitivity and specificity of the different aortic annulus size and aortic root volume measures. The annulus area oversizing of ≥20% resulted in a sensitivity of 100%, specificity of 55.76%, and positive predictive value (PPV) of 8.75%. In patients receiving 26 mm prostheses, the aortic root volume (ARV <13600 mm(3)) provided a better specificity and PPV (79.63 and 18.52%, respectively). A two-step testing procedure considering the area derived average annulus diameter (Darea <23 mm) as a first separating parameter and then the ARV (<13,600 mm(3)) as a further indicator showed the most promising results with the PPV of 31.25%. Regardless of the procedure steps no false negative results were predicted. CONCLUSIONS: Our data show that the ARV provides a better predictive value for correct prosthesis sizing than established annulus measurements, especially in 'borderline' annuli. We suggest a two-step testing procedure for prostheses size selection, considering Darea and ARV to minimize the risk of annulus rupture. Prospective studies and examination of larger datasets are warranted to confirm these findings.

Fibroblast growth factor signaling pathway in endothelial cells is activated by BMPER to promote angiogenesis.

OBJECTIVE: Previously, we have identified bone morphogenetic protein endothelial cell precursor-derived regulator (BMPER) to increase the angiogenic activity of endothelial cells in a concentration-dependent manner. In this project, we now investigate how BMPER acts in concert with key molecules of angiogenesis to promote blood vessel formation. APPROACH AND RESULTS: To assess the effect of BMPER on angiogenesis-related signaling pathways, we performed an angiogenesis antibody array with BMPER-stimulated endothelial cells. We detected increased basic fibroblast growth factor (bFGF/FGF-2) expression after BMPER stimulation and decreased expression of thrombospondin-1. Additionally, FGF receptor-1 expression, phosphorylation, FGF signaling pathway activity, and cell survival were increased. Consistently, silencing of BMPER by small interfering RNA decreased bFGF and FGF receptor-1 expression and increased thrombospondin-1 expression and cell apoptosis. Next, we investigated the interaction of BMPER and the FGF signaling pathway in endothelial cell function. BMPER stimulation increased endothelial cell angiogenic activity in migration, Matrigel, and spheroid assays. To block FGF signaling, an anti-bFGF antibody was used, which effectively inhibited the proangiogenic BMPER effect. Accordingly, BMPER-silenced endothelial cells under bFGF stimulation showed decreased angiogenic activity compared with bFGF control. We confirmed these findings in vivo by subcutaneous Matrigel injections with and without bFGF in C57BL/6_Bmper(+/-) mice. Aortic ring assays of C57BL/6_Bmper(+/-) mice confirmed a specific effect for bFGF but not for vascular endothelial growth factor. CONCLUSIONS: Taken together, the proangiogenic BMPER effect in endothelial cells is mediated by inhibition of antiangiogenic thrombospondin-1 and enhanced expression and activation of the FGF signaling pathway that is crucial in the promotion of angiogenesis.

Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors.

Dabigatran and rivaroxaban are novel, vitamin K-independent oral anticoagulants (NOACs) and act via antagonism of the coagulation factor (F) IIa (dabigatran) or FXa (rivaroxaban), respectively. Compared to vitamin-K-antagonists, NOACs have shown non-inferiority of risk and benefit in patients with non valvular atrial fibrillation (AF). In clinical practice there is increasing use of NOACs combined with platelet inhibitors in patients with AF and coronary artery disease. However, whether NOACs affect the function of platelet inhibitors remains incompletely known. This observational study aimed to assess the platelet function in patients receiving dabigatran or rivaroxaban and concomitant platelet inhibitors. A single centre observational study was performed analysing the platelet aggregation of patients treated with dabigatran or rivaroxaban with or without concomitant platelet inhibitors. Measurements before the initiation of NOAC therapy served as the respective control group. Platelet aggregation was measured by multiple electrode aggregometry and was induced with adenosine diphosphate (ADP, 6.5 µM) and arachidonic acid (AA, 0.5 mM), respectively. In order to evaluate whether NOACs interact with platelet inhibition by ASA or the P2Y12-antagonist clopidogrel, 87 patients were grouped according to their concomitant antiplatelet medication. Comparing the ADP- and AA-induced platelet aggregation in patients without concomitant platelet inhibitors (n = 45) no significant differences under therapy with dabigatran (d) or rivaroxaban (r) compared to the control group (c) were observed. In patients taking clopidogrel as a concomitant platelet inhibitor (n = 21), neither dabigatran nor rivaroxaban affected the ADP-induced platelet aggregation (c 20 ± 11, d 21 ± 14, r 18 ± 8 AU*min, p = 0.200). Patients receiving dabigatran or rivaroxaban in combination with ASA (n = 42; 21 ASA only, 21 ASA + clopidogrel) showed no significant differences of the AA-induced aggregation compared to the control group (c 10 ± 8, d 9 ± 7, r 10 ± 8 AU*min, p = 0.810). The antiplatelet effects of ASA and clopidogrel monitored by AA- or ADP-induced platelet aggregation were not affected by NOAC therapy.

Platelet reactivity after administration of third generation P2Y12-antagonists does not depend on body weight in contrast to clopidogrel.

The current standard of antiplatelet therapy for patients with myocardial infarction (MI) includes the P2Y12-receptor antagonist clopidogrel, prasugrel or ticagrelor. While it has been shown that platelet reactivity after clopidogrel administration depends on factors such as body weight, it is not known if these factors have an effect on the activity of prasugrel or ticagrelor. Thus, this study aimed to analyse factors associated with high residual platelet reactivity after administration of third generation P2Y12-antagonists compared to clopidogrel. In a single centre registry the antiplatelet effect of clopidogrel, prasugrel or ticagrelor was investigated by aggregometry in patients after MI. To assess the overall capacity of platelet aggregation whole blood was induced with thrombin receptor activating peptide (TRAP; 32 µM). To specifically quantify the effect of P2Y12-antagonists, blood was stimulated with 6.4 µM adenosine diphophosphate (ADP). Relative ADP induced aggregation (r-ADP-agg) was defined as the ADP-TRAP-ratio to reflect an individual degree of P2Y12-dependent platelet inhibition. Platelet function of 238 patients was analysed [clopidogrel (n = 58), prasugrel (n = 65), ticagrelor (n = 115)]. It was found that the r-ADP-agg correlated significantly with body weight in patients after clopidogrel administration (r = 0.423; p < 0.001). In contrast, this association was not present in patients after prasugrel (r = -0.117; p = 0.354) or ticagrelor (r = -0.082; p = 0.382) administration. Comparison of the correlation coefficients showed a significant difference (p = 0.003). In contrast to clopidogrel, platelet reactivity after administration of prasugrel or ticagrelor does not depend on body weight in patients after MI. Hence, our mechanistic data support the results of large clinical trials indicating that patients with high body weight do not need to be treated with increased doses of third generation P2Y12-antagonists to achieve sufficient platelet inhibition (registry for patients after myocardial infarction treated with antiplatelet agents; DRKS00003146).

High platelet reactivity after P2Y12-inhibition in patients with atrial fibrillation and coronary stenting.

High platelet reactivity (HPR) after P2Y12-inhibition in patients undergoing coronary stenting is associated with an increased risk for thromboembolic events and coronary death. So far it is not known how HPR affects the clinical outcome of different treatment strategies in patients with atrial fibrillation (AF) undergoing coronary stenting. In this single centre, observational study the antiplatelet effect of P2Y12-inhibitors in AF patients undergoing coronary stenting was investigated using impedance aggregometry. Patients received either dual antiplatelet therapy (DAPT) or triple therapy (TT). HPR was defined as the ratio of ADP-to TRAP-induced aggregation (r-ADP-agg) ≥50 %. Thromboembolic and bleeding events were assessed within the first 30 days after stenting. Out of 910 screened patients 167 patients were available for the present analysis. HPR was found in 5 of 43 (12 %) patients treated with DAPT and in 18 of 124 (15 %) patients treated with TT. In patients receiving TT, HPR was not a risk factor for thromboembolic events compared to patients with adequate response to P2Y12-inhibitors (6 vs. 8 %, p = 0.712). There was a trend for less bleeding events in patients with HPR compared to r-ADP-agg <50 % in the TT group (0 vs. 16 %, p = 0.077). Our data suggest that HPR after P2Y12-antagonism in patients receiving TT due to AF and coronary stenting might protect from bleeding without increasing thromboembolic risk. Future studies will need to investigate if patients with AF receiving coronary stenting benefit from a reduction of antithrombotic therapy.

BMPER-induced BMP signaling promotes coronary artery remodeling.

The connection of the coronary vasculature to the aorta is one of the last essential steps of cardiac development. However, little is known about the signaling events that promote normal coronary artery formation. The bone morphogenetic protein (BMP) signaling pathway regulates multiple aspects of endothelial cell biology but has not been specifically implicated in coronary vascular development. BMP signaling is tightly regulated by numerous factors, including BMP-binding endothelial cell precursor-derived regulator (BMPER), which can both promote and repress BMP signaling activity. In the embryonic heart, BMPER expression is limited to the endothelial cells and the endothelial-derived cushions, suggesting that BMPER may play a role in coronary vascular development. Histological analysis of BMPER(-/-) embryos at early embryonic stages demonstrates that commencement of coronary plexus differentiation is normal and that endothelial apoptosis and cell proliferation are unaffected in BMPER(-/-) embryos compared with wild-type embryos. However, analysis between embryonic days 15.5-17.5 reveals that, in BMPER(-/-) embryos, coronary arteries are either atretic or connected distal to the semilunar valves. In vitro tubulogenesis assays indicate that isolated BMPER(-/-) endothelial cells have impaired tube formation and migratory ability compared with wild-type endothelial cells, suggesting that these defects may lead to the observed coronary artery anomalies seen in BMPER(-/-) embryos. Additionally, recombinant BMPER promotes wild-type ventricular endothelial migration in a dose-dependent manner, with a low concentration promoting and high concentrations inhibiting migration. Together, these results indicate that BMPER-regulated BMP signaling is critical for coronary plexus remodeling and normal coronary artery development.