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Van Der Laan S.W.,Laboratory of Experimental Cardiology | Foroughi Asl H.,Karolinska Institutet | van den Borne P.,Laboratory of Experimental Cardiology | van Setten J.,Laboratory of Experimental Cardiology | And 13 more authors.
Atherosclerosis | Year: 2015

Background: The eicosanoid genes ALOX5, ALOX5AP and LTA4H have been implicated in atherosclerosis. We assessed the impact of common variants in these genes on gene expression, circulating protein levels, and atherosclerotic plaque phenotypes. Methods: We included patients from the Stockholm Atherosclerosis Gene Expression study (STAGE, N=109), and the Athero-Express Biobank Study (AE, N=1443). We tested 1453 single-nucleotide variants (SNVs) in ALOX5, ALOX5AP and LTA4H for association with gene expression in STAGE. We also tested these SNVs for association with seven histologically defined plaque phenotypes in the AE (which included calcification, collagen, cellular content, atheroma size, and intraplaque vessel density and hemorrhage). Results: We replicate a known cis-eQTL (rs6538697, p=1.96×10-6) for LTA4H expression in whole blood of patients from STAGE. We found no significant association for any of the SNVs tested with serum levels of ALOX5 or ALOX5AP (p>5.79×10-4). For atherosclerotic plaque phenotypes the strongest associations were found for intraplaque vessel density and smooth muscle cells in the ALOX5AP locus (p>1.67×10-4). Conclusions: We replicate a known eQTL for LTA4H expression in whole blood using STAGE data. We found no associations of variants in and around ALOX5, ALOX5AP and LTA4H with serum ALOX5 or ALOX5AP levels, or plaque phenotypes. On the supposition that these genes play a causal role in atherosclerosis, these results suggest that common variants in these loci play a limited role (if any) in influencing advanced atherosclerotic plaque morphology to the extent that it impacts atherosclerotic disease. © 2015 Elsevier Ireland Ltd.


Tersteeg C.,Laboratory of Clinical Chemistry and Haematology | Tersteeg C.,Laboratory of Experimental Cardiology | Heijnen H.F.,Laboratory of Clinical Chemistry and Haematology | Heijnen H.F.,Cell Microscopy Center | And 10 more authors.
Circulation Research | Year: 2014

RATIONALE:: Platelets are the most important cells in the primary prevention of blood loss after injury. In addition, platelets are at the interface between circulating leukocytes and the (sub)endothelium regulating inflammatory responses. OBJECTIVE:: Our aim was to study the dynamic process that leads to the formation of procoagulant and proinflammatory platelets under physiological flow. METHODS AND RESULTS:: In the present study, we describe the formation of extremely long, negatively charged membrane strands that emerge from platelets adhered under flow. These flow-induced protrusions (FLIPRs) are formed in vitro on different physiological substrates and are also detected in vivo in a mouse carotid injury model. FLIPRs are formed downstream the adherent and activated platelets and reach lengths of 250 μm. FLIPR formation is shear-dependent and requires cyclophilin D, calpain, and Rac1 activation. It is accompanied by a disassembly of the F-actin and microtubule organization. Monocytes and neutrophils roll over FLIPRs in a P-selectin/P-selectin glycoprotein ligand-1-dependent manner, retrieving fragments of FLIPRs as microparticles on their surface. Consequently, monocytes and neutrophils become activated, as demonstrated by increased CD11b expression and L-selectin shedding. CONCLUSIONS:: The formation of long platelet membrane extensions, such as the ones presented in our flow model, may pave the way to generate an increased membrane surface for interaction with monocytes and neutrophils. Our study provides a mechanistic model for platelet membrane transfer and the generation of monocyte/neutrophil-microparticle complexes. We propose that the formation of FLIPRs in vivo contributes to the well-established proinflammatory function of platelets and platelet-derived microparticles. © 2014 American Heart Association, Inc.


Bot P.T.,AMC | Bot P.T.,Laboratory of Experimental Cardiology | Grundmann S.,Albert Ludwigs University of Freiburg | Goumans M.-J.,UMC | And 10 more authors.
Atherosclerosis | Year: 2011

Objective: Atherosclerosis is an inflammatory disease, modulated by plaque stabilizing and de-stabilizing cell populations such as infiltrating monocytes and vascular smooth muscle cells (vSMCs). Transcription factors regulating proliferation and differentiation of atherosclerosis relevant cell types are of interest in this context. The forkhead box transcription factor FoxP1 modulates monocyte differentiation. We studied FoxP1 expression in atherosclerotic tissue, correlated FoxP1 expression with plaque characteristics and identified associations between FoxP1 and plaque proteins. Methods: 116 Atherosclerotic plaques from carotid endarterectomy samples were histologically classified (fibrous, fibroatheromatous, atheromatous) and subjected to semi-quantitative protein analysis. Macrophage, SMC content and intraplaque thrombus amount were determined histologically. FoxP1 expression was investigated by western blotting and immunohistochemistry. In addition FoxP1 was overexpressed in vitro to identify causal relations between FoxP1 and plaque proteins. Results: FoxP1 expression was observed in SMCs, macrophages, endothelial cells and T-cells within the plaque. High SMC and collagen content correlated with increased FoxP1 isoform (72 kD and 95 kD) levels 72 kD FoxP1 expression was lower in plaques containing intraplaque thrombus. FoxP1 correlated with active intraplaque TGFβ signaling. In vitro stimulation of SMCs with TGFβ resulted in increased FoxP1 levels 72 kD FoxP1 correlated with expression of pro-fibrotic EGR-1 and increased Col1A1 expression. Conclusion: FoxP1 is expressed by different cell types in atherosclerotic lesions and associated with more stable plaque characteristics and intraplaque TGFβ signaling. FoxP1 expression in vitro is induced by TGFβ, resulting in increased collagen and EGR-1 expression, providing a mechanism for the observed association with a more stable plaque phenotype. © 2011 Elsevier Ireland Ltd.


Burgers L.T.,Erasmus University Rotterdam | McClellan E.A.,Metropolitan State University of Denver | Hoefer I.E.,Laboratory of Experimental Cardiology | Pasterkamp G.,Laboratory of Experimental Cardiology | And 9 more authors.
Netherlands Heart Journal | Year: 2016

Aim Variations in treatment are the result of differences in demographic and clinical factors (e.g. anatomy), but physician and hospital factors may also contribute to treatment variation. The choice of treatment is considered important since it could lead to differences in long-term outcomes. This study explores the associations with stent choice: i.e. drug-eluting stent (DES) versus bare-metal stents (BMS) for Dutch patients diagnosed with stable or unstable coronary artery disease (CAD). Methods & results Associations with treatment decisions were based on a prospective cohort of 692 patients with stable or unstable CAD. Of those patients, 442 patients were treated with BMS or DES. Multiple logistic regression analyses were performed to identify variables associated with stent choice. Bivariate analyses showed that NYHA class, number of diseased vessels, previous percutaneous coronary intervention, smoking, diabetes, and the treating hospital were associated with stent type. After correcting for other associations the treating hospital remained significantly associated with stent type in the stable CAD population. Conclusions This study showed that several factors were associated with stent choice. While patients generally appear to receive the most optimal stent given their clinical characteristics, stent choice seems partially determined by the treating hospital, which may lead to differences in longterm outcomes. © The Author(s) 2016.


PubMed | Cardiovascular Research Institute, Karolinska Institutet, Max Delbrück Center for Molecular Medicine, Roslin Institute and 2 more.
Type: Journal Article | Journal: Atherosclerosis | Year: 2015

The eicosanoid genes ALOX5, ALOX5AP and LTA4H have been implicated in atherosclerosis. We assessed the impact of common variants in these genes on gene expression, circulating protein levels, and atherosclerotic plaque phenotypes.We included patients from the Stockholm Atherosclerosis Gene Expression study (STAGE, N = 109), and the Athero-Express Biobank Study (AE, N = 1443). We tested 1453 single-nucleotide variants (SNVs) in ALOX5, ALOX5AP and LTA4H for association with gene expression in STAGE. We also tested these SNVs for association with seven histologically defined plaque phenotypes in the AE (which included calcification, collagen, cellular content, atheroma size, and intraplaque vessel density and hemorrhage).We replicate a known cis-eQTL (rs6538697, p = 1.96 10(-6)) for LTA4H expression in whole blood of patients from STAGE. We found no significant association for any of the SNVs tested with serum levels of ALOX5 or ALOX5AP (p > 5.79 10(-4)). For atherosclerotic plaque phenotypes the strongest associations were found for intraplaque vessel density and smooth muscle cells in the ALOX5AP locus (p > 1.67 10(-4)).We replicate a known eQTL for LTA4H expression in whole blood using STAGE data. We found no associations of variants in and around ALOX5, ALOX5AP and LTA4H with serum ALOX5 or ALOX5AP levels, or plaque phenotypes. On the supposition that these genes play a causal role in atherosclerosis, these results suggest that common variants in these loci play a limited role (if any) in influencing advanced atherosclerotic plaque morphology to the extent that it impacts atherosclerotic disease.


Dries E.,Laboratory of Experimental Cardiology | Bito V.,Laboratory of Experimental Cardiology | Bito V.,Hasselt University | Lenaerts I.,Laboratory of Experimental Cardiology | And 5 more authors.
Circulation Research | Year: 2013

RATIONALE: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. OBJECTIVE: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. METHODS AND RESULTS: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca indicator). Ca sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca/calmodulin-dependent kinase II blockers autocamtide-2-related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca transients, frequency-dependent increase of the rate of Ca rise was seen in coupled RyR regions only and abolished by autocamtide-2-related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. CONCLUSIONS: Coupled RyRs have a distinct modulation by Ca/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca/reactive oxygen species microdomain, and subject to modification with disease. © 2013 American Heart Association, Inc.


Van Holten T.C.,Laboratory for Clinical Chemistry and Haematology | Bleijerveld O.B.,Laboratory of Experimental Cardiology | Bleijerveld O.B.,University Utrecht | Bleijerveld O.B.,Netherlands Proteomics Center | And 10 more authors.
Cardiovascular Research | Year: 2014

Aims Platelets are a natural source of growth factors, cytokines and chemokines, that regulate angiogenesis and inflammation. It has been suggested that differential release of pro- and anti-angiogenic growth factors from platelet α-granules by protease-activated receptors (PAR) 1 and 4 may be important for the regulation of angiogenesis. We aimed to compare the releasates of unstimulated platelets with PAR-1- and PAR-4-stimulated platelets. Methods and results The release of -thromboglobulin, platelet factor (PF)-4, thrombospondin, platelet-derived growth factor (PDGF)-A/B, regulated and normal T-cell expressed and secreted (RANTES/CCL5), endostatin, CXCL12, and vascular endothelial growth factor (VEGF) was measured with enzyme-linked immunosorbent assay (ELISA). Mass spectrometry (MS)-based quantitative proteomics identified 93 proteins from platelets stimulated with PAR-1 and PAR-4. A strong correlation between the factors released after either stimulus was observed (Spearman's r 0.94, P < 0.001). Analysis with ELISA showed that stimulation with PAR-1 or PAR-4 lead to non-differential release of -thromboglobulin, PF-4, thrombospondin, PDGF-A/B, RANTES/CCL5, endostatin, CXCL12, and VEGF. Release of thrombospondin was slightly lower after PAR-1 stimulation (7.2 μg/mL), compared with PAR-4 induced release (9.8 μg/mL; P < 0.05). Conclusions Both ELISA on established α-granule proteins and MS-based quantitative proteomics showed that the most abundant α-granule proteins are released in similar quantities from platelets after stimulation with either PAR-1 or PAR-4. Our findings provide evidence against the hypothesis that PAR-1 and PAR-4 stimulation of platelets trigger differential release of alpha-granule, but further studies are needed to draw conclusions for physiological conditions. © 2014 Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014.


Tersteeg C.,Laboratory of Clinical Chemistry and Haematology | Tersteeg C.,Laboratory of Experimental Cardiology | De Maat S.,Laboratory of Clinical Chemistry and Haematology | De Meyer S.F.,Catholic University of Leuven | And 8 more authors.
Circulation | Year: 2014

BACKGROUND - : Von Willebrand factor (VWF) multimer size is controlled through continuous proteolysis by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13). This prevents spontaneous platelet agglutination and microvascular obstructions. ADAMTS13 deficiency is associated with thrombotic thrombocytopenic purpura, in which life-threatening episodes of microangiopathy damage kidneys, heart, and brain. Enigmatically, a complete ADAMTS13 deficiency does not lead to continuous microangiopathy. We hypothesized that plasmin, the key enzyme of the fibrinolytic system, serves as a physiological backup enzyme for ADAMTS13 in the degradation of pathological platelet-VWF complexes. METHODS AND RESULTS - : Using real-time microscopy, we determined that plasmin rapidly degrades platelet-VWF complexes on endothelial cells in absence of ADAMTS13, after activation by urokinase-type plasminogen activator or the thrombolytic agent streptokinase. Similarly, plasmin degrades platelet-VWF complexes in platelet agglutination studies. Plasminogen directly binds to VWF and its A1 domain in a lysine-dependent manner, as determined by enzyme-linked immunosorbent assay. Plasma levels of plasmin-α2-antiplasmin complexes increase with the extent of thrombocytopenia in patients with acute episodes of thrombotic thrombocytopenic purpura, independent of ADAMTS13 activity. This indicates that plasminogen activation takes place during microangiopathy. Finally, we show that the thrombolytic agent streptokinase has therapeutic value for Adamts13 mice in a model of thrombotic thrombocytopenic purpura. CONCLUSIONS - : We propose that plasminogen activation on endothelial cells acts as a natural backup for ADAMTS13 to degrade obstructive platelet-VWF complexes. Our findings indicate that thrombolytic agents may have therapeutic value in the treatment of microangiopathies and may be useful to bypass inhibitory antibodies against ADAMTS13 that cause thrombotic thrombocytopenic purpura. © 2014 American Heart Association, Inc.


Haarmark C.,Laboratory of Experimental Cardiology | Haarmark C.,Copenhagen University | Kyvik K.O.,Frederiksberg Hospital | Kyvik K.O.,University of Southern Denmark | And 5 more authors.
Circulation: Cardiovascular Genetics | Year: 2011

Background-Tpeak-Tend interval (TpTe) and T-wave amplitude (Tamp) carry diagnostic and prognostic information regarding cardiac morbidity and mortality. Heart rate and QT interval are known to be heritable traits. The heritability of T-wave morphology parameters such as TpTe and Tamp is unknown. TpTe and Tamp were evaluated in a large sample of twins. Methods and Results-Twins from the GEMINAKAR study (611 pairs, 246 monozygotic, 365 dizygotic; mean age, 38-11 years; 49% men) who had an ECG performed during 1997 to 2000 were included. Tamp was measured in leads V1 and V5. Duration variables (RR interval, QTpeak and QTend interval) were measured and averaged over 3 consecutive beats in lead V5. TpTe was calculated as the QTend- and QTpeak-interval difference. Heritability was assessed using structural equation models adjusting for age, sex, and body mass index. All models were reducible to a model of additive genetics and unique environment. All variables had considerable genetic components. Adjusted heritability estimates were as follows: TpTe, 46%; Tamp lead V1, 34%; Tamp lead V5, 47%; RR interval, 55%; QT interval, 67%; and Bazett-corrected QT interval, 42%. Conclusions-RR interval, QT interval, Tamp, and TpTe interval are heritable ECG parameters. © 2011 American Heart Association, Inc.


PubMed | Laboratory of Experimental Cardiology, UMC Utrecht and National University Hospital Singapore
Type: Journal Article | Journal: Cardiovascular research | Year: 2015

Restoration of coronary blood flow is crucial in the treatment of acute myocardial infarction. Reperfusion, however, induces ischaemia-reperfusion (IR) injury, which further deteriorates myocardial function. The innate immune system plays an important role in this process, mediating rapid influx of immune cells into the reperfused myocardium. Leukotriene B4 is an important leucocyte chemoattractant, performing its actions through binding to its specific receptor BLT1. We hypothesized that treatment with LSN2792613, a selective BLT1 antagonist, reduces infarct size (IS) in a mouse model of myocardial IR injury.Male C57Bl/6J mice were subjected to myocardial ischaemia for 30 min by surgical coronary artery ligation, followed by reperfusion. Mice received either LSN2792613 or vehicle, three times daily (orally) for up to 72 h after reperfusion. BLT1 inhibition with LSN2792613 reduced IS compared with vehicle treatment (26.9 2.7 vs. 34.9 2.2%, P = 0.030) at 24 h after reperfusion. The levels of IL-6 and keratinocyte chemoattractant were reduced in the infarcted tissue of LSN2792613-treated mice. Reduced apoptosis in LSN2792613-treated mice was suggested by increased levels of phosphorylated JNK and GSK3/, and confirmed by flow cytometric analysis showing less apoptotic and necrotic cardiomyocytes in the infarcted myocardium. Echocardiography at 4 weeks after myocardial IR showed a slightly higher ejection fraction and stroke volume in mice treated with LSN2792613 compared with vehicle-treated mice, whereas left ventricular volumes were comparable.Selective BLT1 inhibition with LSN2792613 reduces inflammation and apoptosis following IR, resulting in reduced IS, and therefore might be a promising strategy to prevent myocardial IR injury.

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