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1.  Calcification Characteristics of Low-Flow Low-Gradient Severe Aortic Stenosis in Patients Undergoing Transcatheter Aortic Valve Replacement 
Low-flow low-gradient severe aortic stenosis (LFLGAS) is associated with worse outcomes. Aortic valve calcification patterns of LFLGAS as compared to non-LFLGAS have not yet been thoroughly assessed. 137 patients undergoing transcatheter aortic valve replacement (TAVR) with preprocedural multidetector computed tomography (MDCT) and postprocedural transthoracic echocardiography were enrolled. Calcification characteristics were assessed by MDCT both for the total aortic valve and separately for each leaflet. 34 patients had LFLGAS and 103 non-LFLGAS. Total aortic valve calcification volume (p < 0.001), mass (p < 0.001), and density (p = 0.004) were lower in LFLGAS as compared to non-LFLGAS patients. At 30-day follow-up, mean transaortic pressure gradients and more than mild paravalvular regurgitation did not differ between groups. In conclusion, LFLGAS and non-LFLGAS express different calcification patterns which, however, did not impact on device success after TAVR.
PMCID: PMC4576007  PMID: 26435875
2.  Welcome to the ESC journal family 
PMCID: PMC3760551  PMID: 24062882
3.  Anti-inflammatory therapies for cardiovascular disease 
European Heart Journal  2014;35(27):1782-1791.
Atherothrombosis is no longer considered solely a disorder of lipoprotein accumulation in the arterial wall. Rather, the initiation and progression of atherosclerotic lesions is currently understood to have major inflammatory influences that encompass components of both the innate and acquired immune systems. Promising clinical data for ‘upstream’ biomarkers of inflammation such as interleukin-6 (IL-6) as well as ‘downstream’ biomarkers such as C-reactive protein, observations regarding cholesterol crystals as an activator of the IL-1β generating inflammasome, and recent Mendelian randomization data for the IL-6 receptor support the hypothesis that inflammatory mediators of atherosclerosis may converge on the central IL-1, tumour necrosis factor (TNF-α), IL-6 signalling pathway. On this basis, emerging anti-inflammatory approaches to vascular protection can be categorized into two broad groups, those that target the central IL-6 inflammatory signalling pathway and those that do not. Large-scale Phase III trials are now underway with agents that lead to marked reductions in IL-6 and C-reactive protein (such as canakinumab and methotrexate) as well as with agents that impact on diverse non-IL-6-dependent pathways (such as varespladib and darapladib). Both approaches have the potential to benefit patients and reduce vascular events. However, care should be taken when interpreting these trials as outcomes for agents that target IL-6 signalling are unlikely to be informative for therapies that target alternative pathways, and vice versa. As the inflammatory system is redundant, compensatory, and crucial for survival, evaluation of risks as well as benefits must drive the development of agents in this class.
PMCID: PMC4155455  PMID: 24864079
Inflammation; Atherosclerosis; C reactive protein; Interleukin-6; Inflammasome; Methotrexate; Canakinumab; Salsalate; Darapladib; Colchicine
4.  Electrocardiographic features of disease progression in arrhythmogenic right ventricular cardiomyopathy/dysplasia 
Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is considered a progressive cardiomyopathy. However, data on the clinical features of disease progression are limited. The aim of this study was to assess 12-lead surface electrocardiographic (ECG) changes during long-term follow-up, and to compare these findings with echocardiographic data in our large cohort of patients with ARVC/D.
Baseline and follow-up ECGs of 111 patients from three tertiary care centers in Switzerland were systematically analyzed with digital calipers by two blinded observers, and correlated with findings from transthoracic echocardiography.
The median follow-up was 4 years (IQR 1.9–9.2 years). ECG progression was significant for epsilon waves (baseline 14% vs. follow-up 31%, p = 0.01) and QRS duration (111 ms vs. 114 ms, p = 0.04). Six patients with repolarization abnormalities according to the 2010 Task Force Criteria at baseline did not display these criteria at follow-up, whereas in all patients with epsilon waves at baseline these depolarization abnormalities also remained at follow-up. T wave inversions in inferior leads were common (36% of patients at baseline), and were significantly associated with major repolarization abnormalities (p = 0.02), extensive echocardiographic right ventricular involvement (p = 0.04), T wave inversions in lateral precordial leads (p = 0.05), and definite ARVC/D (p = 0.05).
Our data supports the concept that ARVC/D is generally progressive, which can be detected by 12-lead surface ECG. Repolarization abnormalities may disappear during the course of the disease. Furthermore, the presence of T wave inversions in inferior leads is common in ARVC/D.
PMCID: PMC4407546  PMID: 25599583
Arrhythmogenic Right Ventricular; Cardiomyopathy; Dysplasia; Electrocardiography; T wave inversion
5.  Dual Role of Endothelial Nitric Oxide Synthase in Oxidized LDL-Induced, p66Shc-Mediated Oxidative Stress in Cultured Human Endothelial Cells 
PLoS ONE  2014;9(9):e107787.
The aging gene p66Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66Shc activation.
Methods and Results
Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66Shc at Ser-36, resulting in an enhanced production of superoxide anion (O2-). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66Shc phosphorylation, suggesting that basal NO production inhibits O2- production. oxLDL-induced, p66Shc-mediated O2- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66Shc activation and reduced O2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66Shc activation, and reduced O2- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species.
The present study reports that in primary cultured HAEC treated with oxLDL, p66Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.
PMCID: PMC4172699  PMID: 25247687
6.  Feasibility of second-generation bioresorbable vascular scaffold implantation in complex anatomical and clinical scenarios 
Bioresorbable vascular scaffolds (BVS) have become an emerging tool to treat coronary artery disease. However, the current use of BVS is still widely restricted to stable patients and non-complex lesions. In real-world practice patients are far more complex than those with simple type A lesions and the extended use of BVS to complex lesions and high-risk patients needs to be evaluated. Therefore, we sought to investigate the feasibility and performance of BVS in a broad spectrum of patients.
106 patients underwent in total 193 BVS implantations. We assessed the device-related (cardiac death, target vessel myocardial infarction, ischemia-driven target lesion revascularization) and patient-related (all-cause death, any reinfarction and any revascularization) composite outcomes.
90 % of patients (n = 95) had at least one of the following characteristics: >65 years (35 %), ACS (42 %), tortuous vessels (13 %), calcified (17 %) or thrombotic lesions (12 %), lesions defined as AHA type B2/C (42 %), bifurcations (16 %), chronic total occlusions (9 %) or restenosis (14 %). There was no evidence of significant edge dissection, huge thrombus load or incidence of scaffold dislodgement or scaffold disruption in optical coherence tomography pullbacks. Out of 10,157 struts evaluated within 1,117 cross-sections, 302 were classified as malapposed (2.9 %). During a mean follow-up of 147 ± 119 days the rate of device-related events was 2.0 %, whereas patient-related composite events occurred in 6.1 %.
Our results strongly suggest that BVS implantation is feasible in a wide spectrum of patients and complex anatomy of coronary lesions. Long-term outcome of BVS should be further investigated in unrestricted settings in randomized controlled trials.
Electronic supplementary material
The online version of this article (doi:10.1007/s00392-014-0757-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4315475  PMID: 25173111
Bioresorbable vascular scaffold; Optical coherence tomography
7.  Anacetrapib reduces progression of atherosclerosis, mainly by reducing non-HDL-cholesterol, improves lesion stability and adds to the beneficial effects of atorvastatin 
European Heart Journal  2014;36(1):39-48b.
The present study is the first intervention study in a well-established, translational mouse model for hyperlipidaemia and atherosclerosis showing that anacetrapib dose-dependently reduces atherosclerosis development and adds to the anti-atherogenic effects of atorvastatin. This effect is mainly ascribed to the reduction in non-HDL-C despite a remarkable increase in HDL-C and without affecting HDL functionality. In addition, anacetrapib improves lesion stability.
The residual risk that remains after statin treatment supports the addition of other LDL-C-lowering agents and has stimulated the search for secondary treatment targets. Epidemiological studies propose HDL-C as a possible candidate. Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from atheroprotective HDL to atherogenic (V)LDL. The CETP inhibitor anacetrapib decreases (V)LDL-C by ∼15–40% and increases HDL-C by ∼40–140% in clinical trials. We evaluated the effects of a broad dose range of anacetrapib on atherosclerosis and HDL function, and examined possible additive/synergistic effects of anacetrapib on top of atorvastatin in APOE*3Leiden.CETP mice.
Methods and results
Mice were fed a diet without or with ascending dosages of anacetrapib (0.03; 0.3; 3; 30 mg/kg/day), atorvastatin (2.4 mg/kg/day) alone or in combination with anacetrapib (0.3 mg/kg/day) for 21 weeks. Anacetrapib dose-dependently reduced CETP activity (−59 to −100%, P < 0.001), thereby decreasing non-HDL-C (−24 to −45%, P < 0.001) and increasing HDL-C (+30 to +86%, P < 0.001). Anacetrapib dose-dependently reduced the atherosclerotic lesion area (−41 to −92%, P < 0.01) and severity, increased plaque stability index and added to the effects of atorvastatin by further decreasing lesion size (−95%, P < 0.001) and severity. Analysis of covariance showed that both anacetrapib (P < 0.05) and non-HDL-C (P < 0.001), but not HDL-C (P = 0.76), independently determined lesion size.
Anacetrapib dose-dependently reduces atherosclerosis, and adds to the anti-atherogenic effects of atorvastatin, which is mainly ascribed to a reduction in non-HDL-C. In addition, anacetrapib improves lesion stability.
PMCID: PMC4286319  PMID: 25142968
Cholesteryl ester transfer protein; Non-HDL-cholesterol; HDL-cholesterol; HDL function; Atherosclerosis; Anacetrapib; Atorvastatin
8.  Congenital coronary anomalies detected by coronary computed tomography compared to invasive coronary angiography 
As coronary computed tomography angiography (CCTA) has emerged as a non-invasive alternative for evaluation of coronary anatomy with a lower referral threshold than invasive coronary angiography (ICA), the prevalence of coronary anomalies in CCTA may more closely reflect the true prevalence in the general population. Morphological features of coronary anomalies can be evaluated more precisely by CCTA than by ICA, which might lead to a higher identification of congenital coronary anomalies in CCTA compared to ICA.
To evaluate the incidence, clinical and morphological features of the anatomy of patients with coronary anomalies detected either by coronary computed tomography angiography (CCTA) with prospective ECG-triggering or invasive coronary angiography (ICA).
Consecutive patients underwent 64-slice CCTA (n = 1′759) with prospective ECG-triggering or ICA (n = 9′782) and coronary anatomy was evaluated for identification of coronary anomalies to predefined criteria (origin, course and termination) according to international recommendations.
The prevalence of coronary anomalies was 7.9% (n = 138) in CCTA and 2.1% in ICA (n = 203; p < 0.01). The most commonly coronary anomaly detected by CCTA was myocardial bridging 42.8% (n = 59) vs. 21.2% (n = 43); p < 0.01, while with ICA an absent left main trunk was the most observed anomaly 36.0% (n = 73; p < 0.01). In 9.4% (n = 13) of identified coronary anomalies in CCTA 9.4% were potentially serious coronary anaomalies, defined as a course of the coronary artery between aorta and pulmonary artery were identified.
The prevalence of coronary anomalies is substantially higher with CCTA than ICA even after exclusion of patients with myocardial bridging which is more frequently found with CCTA. This suggests that the true prevalence of coronary anomalies in the general population may have been underestimated based on ICA.
PMCID: PMC4118645  PMID: 25004927
Coronary anomalies; Computed coronary tomography angiography; Invasive coronary angiography
9.  Characterization of Pulmonary Vein Dimensions Using High-Definition 64-Slice Computed Tomography prior to Radiofrequency Catheter Ablation for Atrial Fibrillation 
Background. Contrast-enhanced computed tomography is commonly acquired before radiofrequency catheter ablation (RFCA) for atrial fibrillation (AFib) to guide the procedure. We analyzed pulmonary vein (PV) ostial diameter and volumes on a high-definition 64-slice CT (HDCT) scanner in patients with AFib prior to RFCA. Methods and Results. This retrospective study included 50 patients (mean age 60.2 ± 11.4 years, 30 males) undergoing cardiac HDCT scanning before RFCA for drug refractory AFib and 50 age-, BMI-, and sex-matched controls with normal sinus rhythm undergoing HDCT. PV ostial diameter and volume were measured and calculated using a semiautomatic calliper tool. Total ostial PV volume was significantly increased in patients with AFib as compared to controls (P < 0.005). Similarly, total ostial PV diameter was significantly increased in AFib compared to controls (P < 0.001). In AFib, the largest PV volume and diameters were measured in right superior PV (P < 0.05 versus controls). The difference in PV volume between patients and controls was most pronounced in right superior PVs (P = 0.015). Right middle PVs were found more often in patients with AFib (16/50; 32%) than in normal subjects (7/50; 14%). Conclusion. Enlargement of PV ostial area and enlargement of volume are frequent findings in patients with drug refractory AFib. These parameters may add to the risk stratification for AFib recurrence following RFCA.
PMCID: PMC4096392  PMID: 25089213
10.  Alternatively Spliced Tissue Factor Is Not Sufficient for Embryonic Development 
PLoS ONE  2014;9(5):e97793.
Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.
PMCID: PMC4039448  PMID: 24879059
11.  Effects of the high-density lipoprotein mimetic agent CER-001 on coronary atherosclerosis in patients with acute coronary syndromes: a randomized trial† 
European Heart Journal  2014;35(46):3277-3286.
High-density lipoproteins (HDLs) have several potentially protective vascular effects. Most clinical studies of therapies targeting HDL have failed to show benefits vs. placebo.
To investigate the effects of an HDL-mimetic agent on atherosclerosis by intravascular ultrasonography (IVUS) and quantitative coronary angiography (QCA).
Design and setting
A prospective, double-blinded, randomized trial was conducted at 51 centres in the USA, the Netherlands, Canada, and France. Intravascular ultrasonography and QCA were performed to assess coronary atherosclerosis at baseline and 3 (2–5) weeks after the last study infusion.
Five hundred and seven patients were randomized; 417 and 461 had paired IVUS and QCA measurements, respectively.
Patients were randomized to receive 6 weekly infusions of placebo, 3 mg/kg, 6 mg/kg, or 12 mg/kg CER-001.
Main outcome measures
The primary efficacy parameter was the nominal change in the total atheroma volume. Nominal changes in per cent atheroma volume on IVUS and coronary scores on QCA were also pre-specified endpoints.
The nominal change in the total atheroma volume (adjusted means) was −2.71, −3.13, −1.50, and −3.05 mm3 with placebo, CER-001 3 mg/kg, 6 mg/kg, and 12 mg/kg, respectively (primary analysis of 12 mg/kg vs. placebo: P = 0.81). There was also no difference among groups for the nominal change in per cent atheroma volume (0.02, −0.02, 0.01, and 0.19%; nominal P = 0.53 for 12 mg/kg vs. placebo). Change in the coronary artery score was −0.022, −0.036, −0.022, and −0.015 mm (nominal P = 0.25, 0.99, 0.55), and change in the cumulative coronary stenosis score was −0.51, 2.65, 0.71, and −0.77% (compared with placebo, nominal P = 0.85 for 12 mg/kg and nominal P = 0.01 for 3 mg/kg). The number of patients with major cardiovascular events was 10 (8.3%), 16 (13.3%), 17 (13.7%), and 12 (9.8%) in the four groups.
CER-001 infusions did not reduce coronary atherosclerosis on IVUS and QCA when compared with placebo. Whether CER-001 administered in other regimens or to other populations could favourably affect atherosclerosis must await further study.
Name of the trial registry:; Registry's URL:; Trial registration number: NCT01201837.
PMCID: PMC4258222  PMID: 24780501
Atherosclerosis; Coronary disease; High-density lipoproteins; Clinical trial
12.  Two-year outcomes after percutaneous mitral valve repair with the MitraClip system: durability of the procedure and predictors of outcome 
Open Heart  2014;1(1):e000056.
Analyse 2-year outcomes after MitraClip therapy and identify predictors of outcome.
Consecutive patients (n=74) undergoing MitraClip therapy were included in the MitraSWISS registry and followed prospectively.
A reduction of mitral regurgitation (MR) to ≤ mild was achieved in 32 (43%) patients and to moderate in 31 (42%) patients; 16/63 (25%) patients with initially successful treatment developed recurrent moderate to severe or severe MR during the first year and only 1 patient did so during the second year. At 2 years, moderate or less MR was more frequently present in patients with a transmitral mean gradient <3 mm Hg at baseline (73% vs 23%, p < 0.01) and in patients with a left atrial volume index (LAVI) <50 mL/m2 at baseline (86% vs 52%, p=0.03). More than mild MR post MitraClip, N-terminal probrain natriuretic peptide ≥5000 ng/L at baseline, chronic obstructive pulmonary disease (COPD) and chronic kidney disease (CKD) were associated with reduced survival.
A mean transmitral gradient <3 mm Hg at baseline, an LAVI <50 mL/m2, the absence of COPD and CKD, and reduction of MR to less than moderate were associated with favourable outcome. Given a suitable anatomy, such patients may be excellent candidates for MitraClip therapy. Between 1 and 2 years follow-up, clinical and echocardiographic outcomes were stable, suggesting favourable, long-term durability of the device.
PMCID: PMC4195933  PMID: 25332799
13.  The Sirt1 activator SRT3025 provides atheroprotection in Apoe−/− mice by reducing hepatic Pcsk9 secretion and enhancing Ldlr expression 
European Heart Journal  2014;36(1):51-59.
The deacetylase sirtuin 1 (Sirt1) exerts beneficial effects on lipid metabolism, but its roles in plasma LDL-cholesterol regulation and atherosclerosis are controversial. Thus, we applied the pharmacological Sirt1 activator SRT3025 in a mouse model of atherosclerosis and in hepatocyte culture.
Methods and results
Apolipoprotein E-deficient (Apoe−/−) mice were fed a high-cholesterol diet (1.25% w/w) supplemented with SRT3025 (3.18 g kg−1 diet) for 12 weeks. In vitro, the drug activated wild-type Sirt1 protein, but not the activation-resistant Sirt1 mutant; in vivo, it increased deacetylation of hepatic p65 and skeletal muscle Foxo1. SRT3025 treatment decreased plasma levels of LDL-cholesterol and total cholesterol and reduced atherosclerosis. Drug treatment did not change mRNA expression of hepatic LDL receptor (Ldlr) and proprotein convertase subtilisin/kexin type 9 (Pcsk9), but increased their protein expression indicating post-translational effects. Consistent with hepatocyte Ldlr and Pcsk9 accumulation, we found reduced plasma levels of Pcsk9 after pharmacological Sirt1 activation. In vitro administration of SRT3025 to cultured AML12 hepatocytes attenuated Pcsk9 secretion and its binding to Ldlr, thereby reducing Pcsk9-mediated Ldlr degradation and increasing Ldlr expression and LDL uptake. Co-administration of exogenous Pcsk9 with SRT3025 blunted these effects. Sirt1 activation with SRT3025 in Ldlr−/− mice reduced neither plasma Pcsk9, nor LDL-cholesterol levels, nor atherosclerosis.
We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice. Pharmacological activation of Sirt1 appears promising to be tested in patients for its effects on plasma Pcsk9, LDL-cholesterol, and atherosclerosis.
PMCID: PMC4286317  PMID: 24603306
Sirt1; LDL-cholesterol; Pcsk9; LDL receptor; Atherogenesis
14.  Inhibition of Nicotinamide Phosphoribosyltransferase Reduces Neutrophil-Mediated Injury in Myocardial Infarction 
Antioxidants & Redox Signaling  2013;18(6):630-641.
Aims: Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and recent evidence indicates its role in inflammatory processes. Here, we investigated the potential effects of pharmacological Nampt inhibition with FK866 in a mouse myocardial ischemia/reperfusion model. In vivo and ex vivo mouse myocardial ischemia/reperfusion procedures were performed. Results: Treatment with FK866 reduced myocardial infarct size, neutrophil infiltration, and reactive oxygen species (ROS) generation within infarcted hearts in vivo in a mouse model of ischemia and reperfusion. The benefit of FK866 was not shown in the Langendorff model (ex vivo model of working heart without circulating leukocytes), suggesting a direct involvement of these cells in cardiac injury. Sera from FK866-treated mice showed reduced circulating levels of the neutrophil chemoattractant CXCL2 and impaired capacity to prime migration of these cells in vitro. The release of CXCL8 (human homolog of murine chemokine CXCL2) by human peripheral blood mononuclear cells (PBMCs) and Jurkat cells was also reduced by FK866, as well as by sirtuin (SIRT) inhibitors and SIRT6 silencing, implying a pivotal role for this NAD+-dependent deacetylase in the production of this chemokine. Innovation: The pharmacological inhibition of Nampt might represent an effective approach to reduce neutrophilic inflammation- and oxidative stress-mediated tissue damage in early phases of reperfusion after a myocardial infarction. Conclusions: Nampt inhibition appears as a new strategy to dampen CXCL2-induced neutrophil recruitment and thereby reduce neutrophil-mediated tissue injury in mice. Antioxid. Redox Signal. 18, 630–641.
PMCID: PMC3549207  PMID: 22452634
15.  Clonal restriction and predominance of regulatory T cells in coronary thrombi of patients with acute coronary syndromes 
European Heart Journal  2014;36(17):1041-1048.
Regulatory T cells (Treg) exert anti-inflammatory and atheroprotective effects in experimental atherosclerosis. Treg can be induced against specific antigens using immunization strategies associated with clonal restriction. No data exist on Treg in combination with clonal restriction of T cells in patients with acute coronary syndromes (ACS).
Methods and results
Among T cell subsets characterized by flow cytometry, Treg (CD4+ CD25+ CD127low) were twice as frequent in coronary thrombi compared with peripheral blood. Treg prevailed among T cell subsets identified in coronary thrombi. To evaluate clonal restriction, genomic DNA was extracted from coronary thrombi and peripheral blood in order to evaluate T cell receptor (TCR) β chain diversity by means of Multi-N-plex PCR using a primer specific for all TCR β V gene segments and another primer specific for TCR β J gene segments. T cell receptor diversity was reduced in thrombi compared with peripheral blood (intra-individual comparisons in 16 patients) with 8 gene rearrangements in the TCR common in at least 6 out of 16 analysed coronary thrombi. Compared with age-matched healthy controls (n = 16), TCR diversity was also reduced in peripheral blood of patients with ACS; these findings were independent of peripheral T cell numbers.
We provide novel evidence for a perturbed T cell compartment characterized by clonal restriction in peripheral blood and coronary thrombi from patients with ACS. Our findings warrant further studies on Treg as novel therapeutic targets aimed at enhancing this anti-inflammatory component of adaptive immunity in human atherothrombosis.
PMCID: PMC4416137  PMID: 24419807
Acute coronary syndromes; Immunity; T cells
16.  Deletion of Sirt3 does not affect atherosclerosis but accelerates weight gain and impairs rapid metabolic adaptation in LDL receptor knockout mice: implications for cardiovascular risk factor development 
Basic Research in Cardiology  2013;109(1):399.
Sirt3 is a mitochondrial NAD+-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR−/−) and LDLR/Sirt3 double-knockout (Sirt3−/−LDLR−/−) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR−/− mice. Sirt3 deficiency does not affect atherosclerosis in LDLR−/− mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-013-0399-0) contains supplementary material, which is available to authorized users.
PMCID: PMC3898152  PMID: 24370889
SIRTUIN 3; Atherosclerosis; Metabolism; Oxidative stress
17.  A Novel Electrocardiographic Index for the Diagnosis of Diastolic Dysfunction 
PLoS ONE  2013;8(11):e79152.
Although the assessment of diastolic dysfunction (DD) is an integral part of routine cardiologic examinations, little is known about associated electrocardiographic (ECG) changes. Our aim was to investigate a potential role of ECG indices for the recognition of patients with DD.
Methods and Results
ECG parameters correlating with echocardiographic findings of DD were retrospectively assessed in a derivation group of 172 individuals (83 controls with normal diastolic function, 89 patients with DD) and their diagnostic performance was tested in a validation group of 50 controls and 50 patients. The patient group with a DD Grade 1 and 2 showed longer QTc (422±24ms and 434±32ms vs. 409±25ms, p<0.0005) and shorter Tend–P and Tend–Q intervals, reflecting the electrical and mechanical diastole (240±78ms and 276±108ms vs. 373±110ms, p<0.0001; 409±85ms and 447±115ms vs. 526±119ms, p<0.0001). The PQ–interval was significantly longer in the patient group (169±28ms and 171±38ms vs. 153±22ms, p<0.005). After adjusting for possible confounders, a novel index (Tend–P/[PQxAge]) showed a high performance for the recognition of DD, stayed robust in the validation group (sensitivity 82%, specificity 93%, positive predictive value 93%, negative predictive value 82%, accuracy 88%) and proved a substantial added value when combined with the indexed left atrial volume (LAESVI, sensitivity 90%, specificity 92%, positive predictive value 95%, negative predictive value 86%, accuracy 91%).
A novel electrocardiographic index Tend–P/(PQxAge) demonstrates a high diagnostic accuracy for the diagnosis of DD and yields a substantial added value when combined with the LAESVI.
PMCID: PMC3818461  PMID: 24223898
18.  A signature of circulating microRNAs differentiates takotsubo cardiomyopathy from acute myocardial infarction 
European Heart Journal  2013;35(15):999-1006.
Takotsubo cardiomyopathy (TTC) remains a potentially life-threatening disease, which is clinically indistinguishable from acute myocardial infarction (MI). Today, no established biomarkers are available for the early diagnosis of TTC and differentiation from MI. MicroRNAs (miRNAs/miRs) emerge as promising sensitive and specific biomarkers for cardiovascular disease. Thus, we sought to identify circulating miRNAs suitable for diagnosis of acute TTC and for distinguishing TTC from acute MI.
Methods and results
After miRNA profiling, eight miRNAs were selected for verification by real-time quantitative reverse transcription polymerase chain reaction in patients with TTC (n = 36), ST-segment elevation acute myocardial infarction (STEMI, n = 27), and healthy controls (n = 28). We quantitatively confirmed up-regulation of miR-16 and miR-26a in patients with TTC compared with healthy subjects (both, P < 0.001), and up-regulation of miR-16, miR-26a, and let-7f compared with STEMI patients (P < 0.0001, P < 0.05, and P < 0.05, respectively). Consistent with previous publications, cardiac specific miR-1 and miR-133a were up-regulated in STEMI patients compared with healthy controls (both, P < 0.0001). Moreover, miR-133a was substantially increased in patients with STEMI compared with TTC (P < 0.05). A unique signature comprising miR-1, miR-16, miR-26a, and miR-133a differentiated TTC from healthy subjects [area under the curve (AUC) 0.835, 95% CI 0.733–0.937, P < 0.0001] and from STEMI patients (AUC 0.881, 95% CI 0.793–0.968, P < 0.0001). This signature yielded a sensitivity of 74.19% and a specificity of 78.57% for TTC vs. healthy subjects, and a sensitivity of 96.77% and a specificity of 70.37% for TTC vs. STEMI patients. Additionally, we noticed a decrease of the endothelin-1 (ET-1)-regulating miRNA-125a-5p in parallel with a robust increase of ET-1 plasma levels in TTC compared with healthy subjects (P < 0.05).
The present study for the first time describes a signature of four circulating miRNAs as a robust biomarker to distinguish TTC from STEMI patients. The significant up-regulation of these stress- and depression-related miRNAs suggests a close connection of TTC with neuropsychiatric disorders. Moreover, decreased levels of miRNA125a-5p as well as increased plasma levels of its target ET-1 are in line with the microvascular spasm hypothesis of the TTC pathomechanism.
PMCID: PMC3985061  PMID: 24046434
Takotsubo cardiomyopathy; Biomarker; Endothelin-1; MicroRNA
19.  The Assessment of Endothelial Function – From Research into Clinical Practice 
Circulation  2012;126(6):753-767.
The discovery of the endothelium as a crucial organ for the regulation of the vasculature to physiological needs and the recognition of endothelial dysfunction as a key pathological condition - which is associated with most if not all cardiovascular risk factors - led to a tremendous boost of endothelial research in the past 3 decades. Despite the possibility to measure endothelial function in the individual and its widespread use in research, its use as a clinical tool in daily medicine is not established yet. We review the most common methods to assess vascular function in humans and discuss their advantages and disadvantages. Furthermore we give an overview about clinical settings were endothelial function measurements may be valuable in individual patients. Specifically, we provide information why endothelial function is not only a risk marker for cardiovascular risk but may also provides prognostic information beyond commonly used risk scores in primary prevention, and in patients with already established coronary disease.
We conclude, that non-invasive endothelial function measurements provide valuable additional information, however, to ascertain its use for daily clinical practice, future research should determine whether endothelial function can be used to guide treatment in the individual and if this translates into better outcomes.
PMCID: PMC3427943  PMID: 22869857
endothelial function; risk factors; atherosclerosis
20.  Intravenous ferric carboxymaltose in iron-deficient chronic heart failure patients with and without anaemia: a subanalysis of the FAIR-HF trial 
European Journal of Heart Failure  2013;15(11):1267-1276.
Therapy with i.v. iron in patients with chronic heart failure (CHF) and iron deficiency (ID) improves symptoms, functional capacity, and quality of life. We sought to investigate whether these beneficial outcomes are independent of anaemia.
Methods and results
FAIR-HF randomized 459 patients with CHF [NYHA class II or III, LVEF ≤40% (NYHA II) or ≤45% (NYHA III)] and ID to i.v. iron as ferric carboxymaltose (FCM) or placebo in a 2:1 ratio. We analysed the efficacy and safety according to the presence or absence of anaemia (haemoglobin ≤120 g/L) at baseline. Of 459 patients, 232 had anaemia at baseline (51%). The effect of FCM on the primary endpoints of self-reported Patient Global Assessment (PGA) and NYHA class at week 24 was similar in patients with and without anaemia [odds ratio (OR) for improvement, 2.48 vs. 2.60, P = 0.97 for PGA and 1.90 vs. 3.39, P = 0.51 for NYHA). Results were also similar for the secondary endpoints, including PGA and NYHA at weeks 4 and 12, 6 min walk test distance, Kansas City Cardiomyopathy Questionnaire overall score, and European Quality of Life-5 Dimensions Visual Analogue Scale at most time points. Regarding safety, no differences were noticed in the rates of death or first hospitalization between FCM and placebo both in anaemic and in non-anaemic patients.
Treatment of ID with FCM in patients with CHF is equally efficacious and shows a similar favourable safety profile irrespective of anaemia. Iron status should be assessed in symptomatic CHF patients both with and without anaemia and treatment of ID should be considered.
PMCID: PMC3806282  PMID: 23787722
Anaemia; Iron deficiency; Heart failure; Intravenous iron; Ferric carboxymaltose
21.  Vascular lesions induced by renal nerve ablation as assessed by optical coherence tomography: pre- and post-procedural comparison with the Simplicity® catheter system and the EnligHTN™ multi-electrode renal denervation catheter 
European Heart Journal  2013;34(28):2141-2148.
Catheter-based renal nerve ablation (RNA) using radiofrequency energy is a novel treatment for drug-resistant essential hypertension. However, the local endothelial and vascular injury induced by RNA has not been characterized, although this importantly determines the long-term safety of the procedure. Optical coherence tomography (OCT) enables in vivo visualization of morphologic features with a high resolution of 10–15 µm. The objective of this study was to assess the morphological features of the endothelial and vascular injury induced by RNA using OCT.
Methods and results
In a prospective observational study, 32 renal arteries of patients with treatment-resistant hypertension underwent OCT before and after RNA. All pre- and post-procedural OCT pullbacks were evaluated regarding vascular changes such as vasospasm, oedema (notches), dissection, and thrombus formation. Thirty-two renal arteries were evaluated, in which automatic pullbacks were obtained before and after RNA. Vasospasm was observed more often after RNA then before the procedure (0 vs. 42%, P < 0.001). A significant decrease in mean renal artery diameter after RNA was documented both with the EnligHTN™ (4.69 ± 0.73 vs. 4.21 ± 0.87 mm; P < 0.001) and with the Simplicity® catheter (5.04 ± 0.66 vs. 4.57 ± 0.88 mm; P < 0.001). Endothelial-intimal oedema was noted in 96% of cases after RNA. The presence of thrombus formations was significantly higher after the RNA then before ablation (67 vs. 18%, P < 0.001). There was one evidence of arterial dissection after RNA with the Simplicity® catheter, while endothelial and intimal disruptions were noted in two patients with the EnligHTN™ catheter.
Here we show that diffuse renal artery constriction and local tissue damage at the ablation site with oedema and thrombus formation occur after RNA and that OCT visualizes vascular lesions not apparent on angiography. This suggests that dual antiplatelet therapy may be required during RNA.
PMCID: PMC3717310  PMID: 23620498
Optical coherence tomography; Catheter-based renal nerve ablation; Vascular lesions; Simplicity catheter system; EnligHTN multi-electrode renal denervation catheter
22.  Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity 
European Heart Journal  2013;34(45):3515-3524.
Received 22 July 2012; revised 29 January 2013; accepted 4 March 2013
Aldosterone plays a crucial role in cardiovascular disease. ‘Systemic’ inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the ‘endothelial’ MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis.
Methods and results
C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high ‘endogenous’ aldosterone) and in ‘exogenous’ aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR.
Obesity-induced endothelial dysfunction depends on the ‘endothelial’ MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.
PMCID: PMC3844149  PMID: 23594590
Obesity; Endothelial; Aldosterone; Mineralocorticoid receptor
23.  Vascular effects and safety of dalcetrapib in patients with or at risk of coronary heart disease: the dal-VESSEL randomized clinical trial 
European Heart Journal  2012;33(7):857-865.
High-density lipoprotein cholesterol (HDL-C) is inversely associated with cardiovascular (CV) events and thus an attractive therapeutic target. However, in spite of marked elevations in HDL-C, the first cholesterol transport protein (CETP) inhibitor torcetrapib raised blood pressure (BP), impaired endothelial function, and increased CV mortality and morbidity. Dalcetrapib is a novel molecule acting on CETP with a different chemical structure to torcetrapib. As HDL stimulates nitric oxide (NO), suppresses inflammation, and exerts protective CV effects, we investigated the effects of dalcetrapib on endothelial function, blood pressure, inflammatory markers, and lipids in patients with, or at risk of, coronary heart disease (CHD) in a double-blind randomized placebo-controlled trial ( number NCT00655538).
Methods and results
Patients with target low-density lipoprotein cholesterol (LDL-C) levels received dalcetrapib 600 mg/day or placebo for 36 weeks on top of standard therapy (including statins). The primary outcome measures were the change from baseline of flow-mediated dilatation (%FMD) of the right brachial artery after 5 min of cuff occlusion at 12 weeks and the 24 h ambulatory blood pressure monitoring (ABPM) at week 4. Secondary outcomes included change from baseline in FMD after 36 weeks and the change in ABPM at 12 and 36 weeks, changes in HDL-C, LDL-C, triglycerides, CETP activity, as well as standard safety parameters. Four hundred seventy-six patients were randomized. Baseline FMD was 4.1 ± 2.2 and 4.0 ± 2.4% with placebo or dalcetrapib, respectively and did not change significantly from placebo after 12 and 36 weeks (P = 0.1764 and 0.9515, respectively). After 4, 24, and 36 weeks of treatment with dalcetrapib, CETP activity decreased by 51, 53, and 56% (placebo corrected, all P < 0.0001), while at weeks 4, 12, and 36 HDL-C increased by 25, 27, and 31% (placebo corrected, all P < 0.0001). Low-density lipoprotein cholesterol levels did not change. At baseline, ABPM was 125 ± 12/74 ± 8mmHg in the placebo and 128 ± 11/75 ± 7mmHg in the dalcetrapib group (P = 0.3372 and 0.1248, respectively, placebo-corrected change from baseline) and did not change for up to 36 weeks. Biomarkers of inflammation, oxidative stress, and coagulation did not change during follow-up except for Lp-PLA2 mass levels which increased by 17% (placebo corrected). Overall 7 patients given dalcetrapib and 8 patients given placebo experienced at least one pre-specified adjudicated event (11 events with dalcetrapib and 12 events with placebo).
The dal-VESSEL trial has established the tolerability and safety of CETP-inhibition with dalcetrapib in patients with or at risk of CHD. Dalcetrapib reduced CETP activity and increased HDL-C levels without affecting NO-dependent endothelial function, blood pressure, or markers of inflammation and oxidative stress. The dal-OUTCOMES trial (NCT00658515) will show whether dalcetrapib improves outcomes in spite of a lack of effect on endothelial function.
PMCID: PMC3345558  PMID: 22345126
High-density; Lipoprotein; Cholesterol (HDL-C); Torcetrapib; Dalcetrapib
24.  Aortic regurgitation after transcatheter aortic valve implantation: mechanisms and implications 
In recent years, transcatheter aortic valve implantation (TAVI) has become an established treatment option for selected high-risk patients with severe aortic stenosis (AS). Favorable results with regard to both hemodynamics and clinical outcome have been achieved with transcatheter valves.
Aortic regurgitation (AR) remains a major concern after TAVI. Echocardiography is the imaging modality of choice to assess AR in these patients due to its wide accessibility and low cost. Mostly mild residual AR has been observed in up to 70% of patients. However, as even a mild degree of AR has been associated with a decreased survival up to two years after TAVI, accurate evaluation and classification of AR is important. AR in transcatheter valves can be divided into three types according to different pathophysiological mechanisms. Besides the well-known transvalvular and paravalvular forms of regurgitation, a third form termed supra-skirtal has recently been observed. A thorough understanding of AR in transcatheter valves may allow to improve device design and implantation techniques to overcome this complication.
The aim of this review is to provide an overview of the three types of AR after TAVI focussing on the different pathophysiological mechanisms.
PMCID: PMC3839218  PMID: 24282741
Transcatheter aortic valve implantation; aortic regurgitation; echocardiography
25.  Depletion of FOXP3+ regulatory T cells promotes hypercholesterolemia and atherosclerosis  
The Journal of Clinical Investigation  2013;123(3):1323-1334.
Atherosclerosis is a chronic inflammatory disease promoted by hyperlipidemia. Several studies support FOXP3-positive regulatory T cells (Tregs) as inhibitors of atherosclerosis; however, the mechanism underlying this protection remains elusive. To define the role of FOXP3-expressing Tregs in atherosclerosis, we used the DEREG mouse, which expresses the diphtheria toxin (DT) receptor under control of the Treg-specific Foxp3 promoter, allowing for specific ablation of FOXP3+ Tregs. Lethally irradiated, atherosclerosis-prone, low-density lipoprotein receptor–deficient (Ldlr–/–) mice received DEREG bone marrow and were injected with DT to eliminate FOXP3+ Tregs. Depletion of Tregs caused a 2.1-fold increase in atherosclerosis without a concomitant increase in vascular inflammation. These mice also exhibited a 1.7-fold increase in plasma cholesterol and an atherogenic lipoprotein profile with increased levels of VLDL. Clearance of VLDL and chylomicron remnants was hampered, leading to accumulation of cholesterol-rich particles in the circulation. Functional and protein analyses complemented by gene expression array identified reduced protein expression of sortilin-1 in liver and increased plasma enzyme activity of lipoprotein lipase, hepatic lipase, and phospholipid transfer protein as mediators of the altered lipid phenotype. These results demonstrate that FOXP3+ Tregs inhibit atherosclerosis by modulating lipoprotein metabolism.
PMCID: PMC3582120  PMID: 23426179

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