Stefanie Jellinghaus

High rates of prasugrel and ticagrelor non-responder in patients treated with therapeutic hypothermia after cardiac arrest.

INTRODUCTION After cardiac arrest due to acute coronary syndromes (ACS) therapeutic hypothermia (HT) is the standard care to reduce neurologic damage. Additionally, the concomitant medical treatment with aspirin and a P2Y12 receptor inhibitor like clopidogrel (Cl), prasugrel (Pr) or ticagrelor (Ti) is mandatory. The platelet inhibitory effect of these drugs under hypothermia remains unclear. METHODS 164 patients with ACS were prospectively enrolled in this study. 84 patients were treated with HT, 80 patients were under normothermia (NT). All patients were treated with aspirin and one of the P2Y12 receptor inhibitors Cl, Pr or Ti. 24h after the initial loading dose the platelet reactivity index (PRI/VASP-index) was determined to achieve the platelet inhibitory effect. RESULTS In the HT-group the PRI/VASP-index was significantly higher compared to the NT-group (54.86%±25.1 vs. 28.98%±22.8; p<0.001). In patients under HT receiving Cl, the platelet inhibition was most markedly reduced (HT vs. NT: 66.39%±19.1 vs. 33.36%±22.1; p<0.001) compared to Pr (HT vs. NT: 37.6%±25.0 vs. 27.04%±25.5; p=0.143) and Ti (HT vs. NT: 41.5%±21.0 vs. 17.83%±14.5; p=0.009). The rate of non-responder defined as PRI/VASP-index>50% was increased in HT compared to NT (60.7% vs. 22.5%; p<0.001) with the highest rates in the group receiving Cl (CL: 82% vs. 26%, p<0.001; Pr: 32% vs. 23%; n.s.; Ti: 30% vs. 8%, n.s.). CONCLUSION The platelet inhibitory effect in patients treated with HT after cardiac arrest is significantly reduced. This effect was most marked with the use of Cl. The new P2Y12-inhibitors Pr and Ti improved platelet inhibition in HT, but could not completely prevent non-responsiveness.

Ephrin-A1/EphA4-mediated adhesion of monocytes to endothelial cells

The Eph receptors represent the largest family of receptor tyrosine kinases. Both Eph receptors and their ephrin ligands are cell-surface proteins, and they typically mediate cell-to-cell communication by interacting at sites of intercellular contact. The major aim of the present study was to investigate the involvement of EphA4-ephrin-A1 interaction in monocyte adhesion to endothelial cells, as this process is a crucial step during the initiation and progression of the atherosclerotic plaque. Immunohistochemical analysis of human atherosclerotic plaques revealed expression of EphA4 receptor and ephrin-A1 ligand in major cell types within the plaque. Short-time stimulation of endothelial cells with the soluble ligand ephrin-A1 leads to a fourfold increase in adhesion of human monocytes to endothelial cells. In addition, ephrin-A1 further increases monocyte adhesion to already inflamed endothelial cells. EphrinA1 mediates its effect on monocyte adhesion via the activated receptor EphA4. This ephrinA1/EphA4 induced process involves the activation of the Rho signaling pathway and does not require active transcription. Rho activation downstream of EphA4 leads to increased polymerization of actin filaments in endothelial cells. This process was shown to be crucial for the proadhesive effect of ephrin-A1. The results of the present study show that ephrin-A1-induced EphA4 forward signaling promotes monocyte adhesion to endothelial cells via activation of RhoA and subsequent stress-fiber formation by a non-transcriptional mechanism.

TNF-α-mediated adhesion of monocytes to endothelial cells—The role of ephrinA1

The ligand ephrin A1 is more often discussed to play a role in the development of the atherosclerotic plaque and in this context especially in the monocyte adhesion to endothelial cells. As tumor necrosis factor-α (TNF-α) is known to induce monocyte adhesion to endothelium and ephrin A1 expression, the present study focuses on the involvement of ephrin A1 in TNF-α-mediated monocyte adhesion. The analysis of different members of the Eph/ephrin system in TNF-α-treated human umbilical vein endothelial cells (HUVEC) revealed that especially ephrinA1 was found to be highly regulated by TNF-α compared to other members of the Eph family. This effect is also present in arterial endothelial cells from the umbilical artery and from the coronary artery. This regulation is dependent on NFκB-activation as shown by the expression of a constitutive-active IκB-mutant. By using siRNA-mediated silencing and adenoviral overexpression of ephrinA1 in HUVEC, the involvement of ephrinA1 in the TNF-α triggered monocyte adhesion to endothelial cells could be demonstrated. In addition, these results could be verified by quantitative adhesion measurement using atomic force microscopy-based single-cell force spectroscopy and under flow conditions. Furthermore, this effect is mediated via the EphA4 receptor. EphrinA1 does not influence the mRNA or protein expression of the adhesion receptors VCAM-1 and ICAM-1 in endothelial cells. However, the surface presentation of these adhesion receptors is modulated in an ephrinA1-dependent manner. In conclusion, these data demonstrate that ephrinA1 plays an important role in the TNF-α-mediated adhesion of monocytes to endothelial cells, which might be of great importance in the context of atherosclerosis.

Intra-aortic balloon pump (IABP) counterpulsation improves cerebral perfusion in patients with decreased left ventricular function

Background: The current goal of treatment after acute ischemic stroke is the increase of cerebral blood flow (CBF) in ischemic brain tissue. Intra-aortic balloon pump (IABP) counterpulsation in the setting of cardiogenic shock is able to reduce left ventricular afterload and increase coronary blood flow. The effects of an IABP on CBF have not been sufficiently examined. We hypothesize that the use of an IABP especially enhances cerebral blood flow in patients with pre-existing heart failure. Methods: In this pilot study, 36 subjects were examined to investigate the effect of an IABP on middle cerebral artery (MCA) transcranial Doppler (TCD) flow velocity change and relative CBF augmentation by determining velocity time integral changes (ΔVTI) in a constant caliber of the MCA compared to a baseline measurement without an IABP. Subjects were divided into two groups according to their left ventricular ejection fraction (LVEF): Group 1 LVEF >30% and Group 2 LVEF ≤30%. Results: Both groups showed an increase in CBF using an IABP. Patients with a LVEF ≤30% showed a significantly higher increase of ΔVTI in the MCA under IABP augmentation compared to patients with a LVEF >30% (20.9% ± 3.9% Group 2 vs.10.5% ± 2.2% Group 1, p<0,05). The mean arterial pressure (MAP) increased only marginally in both groups under IABP augmentation. Conclusions: IABP improves cerebral blood flow, particularly in patients with pre-existing heart failure and highly impaired LVEF. Hence, an IABP might be a treatment option to improve cerebral perfusion in selected patients with cerebral misperfusion and simultaneously existing severe heart failure.

EphrinB2/EphA4-mediated activation of endothelial cells increases monocyte adhesion

The membrane anchored ligand ephrinB2 belongs to the broad Eph/ephrin system and is able to activate different Eph receptors. The Eph receptors belong to the huge group of receptor-tyrosine kinases. Eph receptors as well as their corresponding ephrin ligands are cell-membrane attached proteins. Therefore, direct cell-cell contact is essentially for interaction. It is known that ephrinB2 plays a pivotal role in developmental and in tumour angiogenesis. Previous studies point to a crucial role of the EphA4-receptor in the process of monocyte adhesion. Since ephrinB2 is known as an interaction partner of EphA4, the aim of the present study was to investigate a possible interplay of EphA4-receptor with ephrinB2 during monocyte adhesion to the endothelium. As verified by bulk adhesion assays and atomic-force microscopy based single-cell force spectroscopy, temporary stimulation of endothelial cells from different sources with the soluble ligand ephrinB2 increased monocyte adhesion to endothelial cells. The proadhesive effect of ephrinB2 was independent of an active transcription, but is mediated via the Rho signaling pathway with subsequent modulation of the actin cytoskeleton. Furthermore, ephrinB2 mediated its impact on monocyte adhesion via the receptor EphA4 as shown by siRNA-mediated silencing. Interestingly, ephrinB2 was induced by TNF-α treatment. Silencing of ephrinB2 led to a lowering of the TNF-α mediated monocyte adhesion to endothelial cells. Furthermore, immunohistochemical staining of human atherosclerotic plaque revealed expression of ephrinB2 in macrophages. The results of the present study point to a crucial role of ephrinB2 induced EphA4 forward signaling in the context of monocyte adhesion to endothelial cells. This transcription-independent effect is mediated by Rho signaling induced actin-filament polymerization.

Three-Dimensional Left Ventricular Torsion in Patients With Dilated Cardiomyopathy - A Marker of Disease Severity -

BACKGROUND LV twist has a key role in maintaining left ventricular (LV) contractility during exercise. The purpose of this study was to investigate LV torsion instead of twist as a surrogate marker of peak oxygen uptake (peak V̇O2) assessed by cardiopulmonary exercise testing (CPET) in patients with non-ischemic dilated cardiomyopathy (DCM).Methods and Results:We evaluated 45 outpatients with DCM (50±12 years, 24% females) with 3D speckle-tracking electrocardiography prior to CPET. LV torsion, LV ejection fraction (EF), LV diastolic function, LV global longitudinal (GLS) and circumferential (GCS) strain were quantified. A reduced functional capacity (FC) was defined as a peak V̇O2<20 mL/kg/min. LV torsion correlated most strongly with peak V̇O2(r=0.76, P<0.001). LV torsion instead of twist was an independent predictor of peak V̇O2(B: 0.59 to 0.71, P<0.001) in multivariable analyses. Impaired LV torsion <0.61 degrees/cm was able to predict a reduced FC with higher sensitivity and specificity (0.91 and 0.81; area under the curve (AUC): 0.88, P<0.001) than LV EF, GLS or GCS (AUC 0.64, 0.63 and 0.66; P<0.05 for differences in AUC). CONCLUSIONS Peak V̇O2correlated more strongly with LV torsion than with LV diastolic function, LV EF, GLS or GCS. LV torsion had high accuracy in identifying patients with a reduced FC.

Regulation of endothelial migration and proliferation by ephrin-A1

Endothelial migration and proliferation are fundamental processes in angiogenesis and wound healing of injured or inflamed vessels. The present study aimed to investigate the regulation of the Eph/ephrin-system during endothelial proliferation and the impact of the ligand ephrin-A1 on proliferation and migration of human umbilical venous (HUVEC) and arterial endothelial cells (HUAEC). Endothelial cells that underwent contact inhibition showed a massive induction of ephrin-A1. In contrast, an injury to a confluent endothelial layer, associated with induction of migration and proliferation, showed reduced ephrin-A1 levels. In addition, reducing ephrin-A1 expression by siRNA led to increased proliferation, whereas the overexpression of ephrin-A1 led to decreased proliferative activity. Due to the fact that wound healing is a combination of proliferation and migration, migration was investigated in detail. First, classical wound-healing assays showed increased wound closure in both ephrin-A1 silenced and overexpressing cells. Live-cell imaging enlightened the underlying differences. Silencing of ephrin-A1 led to a faster but more disorientated migration. In contrast, ephrin-A1 overexpression did not influence velocity of the cells, but the migration was more directed in comparison to the controls. Additional analysis of EphA2-silenced cells showed similar results in terms of proliferation and migration compared to ephrin-A1 silenced cells. Detailed analysis of EphA2 phosphorylation on ligand-dependent phospho-site (Y588) and autonomous activation site (S897) revealed a distinct phosphorylation pattern. Furthermore, the endothelial cells ceased to migrate when they came in contact with an ephrin-A1 coated surface. Using a baculoviral-mediated expression system, ephrin-A1 silencing and overexpression was shown to modulate the formation of focal adhesions. This implicates that ephrin-A1 is involved in changes of the actin cytoskeleton which explains the alterations in migratory actions, at least in part. In conclusion, ephrin-A1 expression is regulated by cellular density and is itself a critical determinant of endothelial proliferation. According to current knowledge, ephrin-A1 seems to be remarkably involved in elementary processes of endothelial migration like cellular polarization, migratory direction and speed. These data support the notion that ephrin-A1 plays a pivotal role in basal mechanisms of re-endothelialization.

Atheroprotective role of Caveolin-1 and eNOS in an innovative transplantation model is mainly mediated by local effects

Endothelial dysfunction is crucial in the initiation of atherosclerosis, which is associated with a lack of nitric oxide. The endothelial NO synthase (eNOS) is responsible for constitutive synthesis of NO and inhibited by caveolin-1 (Cav1). In the current study, we examined the influence on intima formation through single and combined deletion of eNOS and Cav1 with a focus on differentiation of local and systemic effects. A sex-mismatch transplantation of denudated aortae from female C57BL/6n (WT), Cav1-/-, eNOS-/- and Cav1-/-/eNOS-/- (C/e--/--) mice in common carotid artery of male WT mice was performed. After six weeks on Western-type diet, the aortae were explanted and intimal lesions were quantified by determining the intima-media-ratio (IMR). Significantly larger plaques were observed in all knockout mice compared to WT. The highest IMR was detected in Cav1-/- arteries associated with an increased expression of α-smooth muscle actin (αSMA) and the proliferating cell nuclear antigen (PCNA). Both were reduced in aortae from C/e--/--. Galectin-3 (Gal3) immunostaining revealed only small infiltrations of macrophages. Systemic cell invasion was detected by Y chromosome fluorescence in situ hybridization (Y-FISH), which showed only small numbers of systemic cells and no differences between the genotypes. Loss of Cav1 increased vascular lesion by enhancing neointimal proliferation. The combined loss of Cav1 and eNOS, compared to Cav1-/-, lowered intima formation, suggesting an increasing effect of eNOS in the absence of Cav1 on vascular lesion. Furthermore, these effects seem to be mediated by local cells rather than by systemically invaded ones.

Comparison of diverse platelet activation markers as indicators for left atrial thrombus in atrial fibrillation

Abstract Atrial fibrillation (AF) is well known for being a major risk factor of thromboembolic stroke. We could recently demonstrate an association of monocyte–platelet aggregates (MPAs) with the degree of thrombogenicity in patients with AF. This study investigated platelet activation markers, as potential biomarkers for the presence of left atrial (LA) thrombus in patients with AF. One hundred and eight patients with symptomatic AF underwent transesophageal echocardiography (TEE) before scheduled cardioversion or pulmonary vein isolation. In order to determine the content of MPAs by flow-cytometric quantification analyses, blood was drawn on the day of TEE. The soluble CD40 Ligand (sCD40L) and soluble P-selectin (sP-selectin) were obtained by Cytometric Bead Arrays (CBA). D-dimer levels were detected by quantitative immunological determination of fibrin degradation products. Clinical, laboratory, and echocardiographic standard parameters were obtained from all patients, including the determination of the flow in the left atrial appendage (LAA). Patients with detected LA thrombus (n = 28) compared with patients without thrombus (n = 80) showed an increased number of common risk factors, such as age, diabetes, heart failure, and coronary artery disease (CAD). The presence of LA thrombus was associated with significantly increased levels of MPAs (147 ± 12 vs. 304 ± 29 per µl; p < 0.00), sCD40L (106.3 ± 31.0 vs. 33.5 ± 2.1 pg/ml, p = 0.027), and D-dimer (0.13 ± 0.02 vs. 0.69 ± 0.21 mg FEU/l, p = 0.015). In contrast, sP-selectin showed no association with LA thrombus. A multivariate regression analysis showed that MPAs, sCD40L as well as D-dimers were independent indicators for the existence of LA thrombus. MPAs above 170 cells/µl indicated LA thrombus with a high sensitivity of 93% and a specificity of 73% (OR 62, 95% CI. 6.9–557.2, p < 0.001) in patients with AF, whereas the D-dimer lost their quality as independent indicator by using the conventional cut-off of 0.5 mg/l within the regression analysis. MPAs, as well as the D-dimer, correlated significantly negatively with the flow in the LAA measured during TEE. The content of MPAs, sCD40L, and D-dimer, but not sP-selectin showed an increased dependence on LA thrombus in patients with AF. In our study group, MPAs showed the best diagnostic test accuracy of the compared platelet markers. The different results of the examined platelet activation markers could be an indication of diverse mechanisms of LA thrombus in AF. Further studies should evaluate whether determination of MPAs in clinical routine may suffice to indicate the presence of LA thrombus in patients with AF.

Repetitive squatting exercise as a diagnostic predictor in patients with dilated cardiomyopathy

Objectives Non-ischaemic dilated cardiomyopathy (DCM) is characterised by a highly variable disease progression. Stress echocardiography and cardiopulmonary exercise testing (CPET) are beneficial in risk assessment, but are labour intensive. Repetitive squatting and standing without weights is a simple exercise (EX). The aim of this study was to investigate the prognostic role of left ventricular (LV) contractile recruitment (CR) after a simple EX of repetitive squatting through three-dimensional (3D) echocardiography. Methods Patients with DCM (LV ejection fraction (EF)<50%, n=68) and age-matched healthy volunteers (n=25) received a 3D echocardiographic evaluation of LV EF before and after 30 repetitions of squatting-standing EX. CR was defined by the change of LV EF (Δ>4%). Patients were followed up prospectively (2 years) for cardiac death and deteriorating heart failure. Results During follow-up, 14 cardiac events occurred (21%) with six deaths and eight severe heart failure deteriorations. A poor CR after squatting EX differentiated DCM patients with cardiac events during follow-up as accurately as a reduced peak oxygen consumption (peak VO 2<20 mL/kg/min) (sensitivity: 0.97 and 0.95). Both had a significant incremental diagnostic value over clinical (age, dyspnoea and natriuretic peptide level) or resting echocardiographic parameters (E/E’ ratio, LV EF and end-diastolic LV volume) to predict cardiac events (global χ2: 16.0 vs 5.3; 19.5 vs 6.1; P<0.01 for all). Conclusions The presence of LV CR after EX of repetitive squatting without weights can stratify risk and predict cardiac events in patients with DCM as correct as CPET.