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ABSTRACT: Statins have beneficial vascular effects beyond their cholesterol-lowering action. Since macrophages play a central role in atherogenesis, we characterized the effects of simvastatin on gene expression profile of human peripheral blood monocyte (HPBM)-macrophages.
Gene expression profile was studied using Affymetrix gene chip analysis. Lentiviral gene transfer of Kruppel-like factor 2 (KLF-2) was used to further study its role in macrophages. Simvastatin treatment lead to downregulation of many pro-inflammatory genes including several chemokines [e.g. monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory proteins-1alpha and beta, interleukin-2 receptor-beta], members of the tumour necrosis factor family (e.g. lymphotoxin beta), vascular cell adhesion molecule-1, and tissue factor (TF). Simvastatin also modulated the expression of several transcription factors essential for inflammation: NF-kappaB relA/p65 subunit and ets-1 were downregulated, and an atheroprotective transcription factor KLF-2 was upregulated. The effects of simvastatin on MCP-1 and TF could be mimicked by KLF-2 overexpression using lentiviral gene transfer.
Simvastatin has a strong anti-inflammatory effect on HPBM cells including upregulation of the atheroprotective factor KLF-2. This may partly explain the beneficial effects of statins on cardiovascular diseases.
Cardiovascular Research 05/2008; 78(1):175-84. · 6.06 Impact Factor
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Beerend P Hierck,
Kim Van der Heiden,
Fanneke E Alkemade,
Simone Van de Pas, Johannes V Van Thienen,
Bianca C W Groenendijk,
Wilhelmina H Bax,
Arnoud Van der Laarse,
Marco C Deruiter,
Anton J G Horrevoets,
Robert E Poelmann
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ABSTRACT: Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Non-ciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krüppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress.
Developmental Dynamics 04/2008; 237(3):725-35. · 2.54 Impact Factor
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Beerend P. Hierck,
Kim Van der Heiden,
Fanneke E. Alkemade,
Simone Van de Pas, Johannes V. Van Thienen,
Bianca C.W. Groenendijk,
Wilhelmina H. Bax,
Arnoud Van der Laarse,
Marco C. DeRuiter,
Anton J.G. Horrevoets,
Robert E. Poelmann
[show abstract]
[hide abstract]
ABSTRACT: Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Non-ciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krüppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress. Developmental Dynamics 237:725–735, 2008. © 2008 Wiley-Liss, Inc.
Developmental Dynamics 02/2008; 237(3):725 - 735. · 2.54 Impact Factor
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ABSTRACT: The purpose of this study was to investigate the presence and functionality of P-selectin glycoprotein ligand-1 (PSGL-1) on activated endothelial cells (ECs).
We show here that PSGL-1 is expressed at the mRNA and protein levels in umbilical vein and microvascular ECs. Furthermore, this endothelial PSGL-1 (ePSGL-1) is functional and mediates adhesion of monocytes or platelet-monocyte complexes (PMCs) to the activated endothelium in a flow model. ePSGL-1 expression was not affected by treating ECs with inflammatory stimuli (tumor necrosis factor alpha, interleukin-1beta, thrombin, or histamine). However, the functional binding capacity of ePSGL-1 to monocytes or P-selectin/Fc chimera significantly increased by stimulation of the ECs with TNFalpha. By means of a siRNA approach to specifically knock-down the genes involved in the glycosylation of PSGL-1 we could show that tumor necrosis factor alpha-induced glycosylation of ePSGL-1 is critical for its binding capacity.
Our results show that ECs express functional PSGL-1 which mediates tethering and firm adhesion of monocytes and platelets to inflamed endothelium.
Arteriosclerosis Thrombosis and Vascular Biology 06/2007; 27(5):1023-9. · 6.37 Impact Factor
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Joost O Fledderus, Johannes V van Thienen,
Reinier A Boon,
Rob J Dekker,
Jakub Rohlena,
Oscar L Volger,
Ann-Pascale J J Bijnens,
Mat J A P Daemen,
Johan Kuiper,
Theo J C van Berkel,
Hans Pannekoek,
Anton J G Horrevoets
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ABSTRACT: Absence of shear stress due to disturbed blood flow at arterial bifurcations and curvatures leads to endothelial dysfunction and proinflammatory gene expression, ultimately resulting in atherogenesis. KLF2 has recently been implicated as a transcription factor involved in mediating the anti-inflammatory effects of flow. We investigated the effect of shear on basal and TNF-alpha-induced genomewide expression profiles of human umbilical vein endothelial cells (HUVECs). Cluster analysis confirmed that shear stress induces expression of protective genes including KLF2, eNOS, and thrombomodulin, whereas basal expression of TNF-alpha-responsive genes was moderately decreased. Promoter analysis of these genes showed enrichment of binding sites for ATF transcription factors, whereas TNF-alpha-induced gene expression was mostly NF-kappaB dependent. Furthermore, human endothelial cells overlying atherosclerotic plaques had increased amounts of phosphorylated nuclear ATF2 compared with endothelium at unaffected sites. In HUVECs, a dramatic reduction of nuclear binding activity of ATF2 was observed under shear and appeared to be KLF2 dependent. Reduction of ATF2 with siRNA potently suppressed basal proinflammatory gene expression under no-flow conditions. In conclusion, we demonstrate that shear stress and KLF2 inhibit nuclear activity of ATF2, providing a potential mechanism by which endothelial cells exposed to laminar flow are protected from basal proinflammatory, atherogenic gene expression.
Blood 06/2007; 109(10):4249-57. · 9.90 Impact Factor
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ABSTRACT: The transcription factor KLF2 is considered an important mediator of the anti-inflammatory and anti-thrombotic properties of the endothelium. KLF2 is absent from low-shear, atherosclerosis-prone sites of the vascular tree but is induced by HMG-CoA reductase inhibitors (statins) in vitro. We studied KLF2-dependent induction of important determinants of the atheroprotective status of the endothelium to determine whether pharmacological intervention, e.g. by statins, can potentially replace shear stress.
Shear stress and statin effects in combination with TNF-alpha were determined in human umbilical vein endothelial cells by quantitative measurements of the steady-state levels and stability of mRNA for KLF2 and its downstream target genes thrombomodulin (TM) and endothelial nitric oxide synthase (eNOS).
We demonstrate that prolonged shear stress has a potential that is superior to that of statins to induce the KLF2-dependent expression of eNOS and TM, especially in the presence of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). These effects can be attributed to the sustained stabilization of KLF2 mRNA by shear, leading to an increased KLF2 protein expression and concomitant strong induction of KLF2 downstream targets. The stabilization of KLF2 mRNA is demonstrated to be dependent on signaling involving phosphoinositide 3-kinase (PI3K).
The stabilization of KLF2 steady-state levels, as induced by prolonged shear stress but not by statins, may be essential for sustaining the quiescent, atheroprotective status of the vascular endothelium under inflammatory conditions.
Cardiovascular Research 12/2006; 72(2):231-40. · 6.06 Impact Factor
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Peter I Bonta,
Claudia M van Tiel,
Mariska Vos,
Thijs W H Pols, Johannes V van Thienen,
Valérie Ferreira,
E Karin Arkenbout,
Jurgen Seppen,
C Arnold Spek,
Tom van der Poll,
Hans Pannekoek,
Carlie J M de Vries
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ABSTRACT: Atherosclerosis is an inflammatory disease in which macrophage activation and lipid loading play a crucial role. In this study, we investigated expression and function of the NR4A nuclear receptor family, comprising Nur77 (NR4A1, TR3), Nurr1 (NR4A2), and NOR-1 (NR4A3) in human macrophages.
Nur77, Nurr1, and NOR-1 are expressed in early and advanced human atherosclerotic lesion macrophages primarily in areas of plaque activation/progression as detected by in situ-hybridization and immunohistochemistry. Protein expression localizes to the nucleus. Primary and THP-1 macrophages transiently express NR4A-factors in response to lipopolysaccharide and tumor necrosis factor alpha. Lentiviral overexpression of Nur77, Nurr1, or NOR-1 reduces expression and production of interleukin (IL)-1beta and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1alpha and -1beta and monocyte chemoattractant protein-1 chemokines. In addition, NR4A-factors reduce oxidized-low-density lipoprotein uptake, consistent with downregulation of scavenger receptor-A, CD36, and CD11b macrophage marker genes. Knockdown of Nur77 or NOR-1 with gene-specific lentiviral short-hairpin RNAs resulted in enhanced cytokine and chemokine synthesis, increased lipid loading, and augmented CD11b expression, demonstrating endogenous NR4A-factors to inhibit macrophage activation, foam-cell formation, and differentiation.
NR4A-factors are expressed in human atherosclerotic lesion macrophages and reduce human macrophage lipid loading and inflammatory responses, providing further evidence for a protective role of NR4A-factors in atherogenesis.
Arteriosclerosis Thrombosis and Vascular Biology 11/2006; 26(10):2288-94. · 6.37 Impact Factor
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Rob J Dekker, Johannes V van Thienen,
Jakub Rohlena,
Saskia C de Jager,
Yvonne W Elderkamp,
Jurgen Seppen,
Carlie J M de Vries,
Erik A L Biessen,
Theo J C van Berkel,
Hans Pannekoek,
Anton J G Horrevoets
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ABSTRACT: Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow.
American Journal Of Pathology 09/2005; 167(2):609-18. · 4.89 Impact Factor
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Gazi S Hossain, Johannes V van Thienen,
Geoff H Werstuck,
Ji Zhou,
Sudesh K Sood,
Jeffrey G Dickhout,
A B Lawrence de Koning,
Damu Tang,
Dongcheng Wu,
Erling Falk,
Ranjana Poddar,
Donald W Jacobsen,
Kezhong Zhang,
Randal J Kaufman,
Richard C Austin
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ABSTRACT: Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and accelerates atherosclerosis in apoE-/- mice. Despite the observations that homocysteine causes endoplasmic reticulum (ER) stress and programmed cell death (PCD) in cultured human vascular endothelial cells, the cellular factors responsible for this effect and their relevance to atherogenesis have not been completely elucidated. We report here that homocysteine induces the expression of T-cell death-associated gene 51 (TDAG51), a member of the pleckstrin homology-related domain family, in cultured human vascular endothelial cells. This effect was observed for other ER stress-inducing agents, including dithiothreitol and tunicamycin. TDAG51 expression was attenuated in homozygous A/A mutant eukaryotic translation initiation factor 2 alpha mouse embryonic fibroblasts treated with homocysteine or tunicamycin, suggesting that ER stress-induced phosphorylation of eukaryotic translation initiation factor 2 alpha is required for TDAG51 transcriptional activation. Transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion, and promoted detachment-mediated PCD. In support of these in vitro findings, TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apoE-/- mice fed hyperhomocysteinemic diets, compared with mice fed a control diet. Collectively, these findings provide evidence that TDAG51 is induced by homocysteine, promotes detachment-mediated PCD, and contributes to the development of atherosclerosis observed in hyperhomocysteinemia.
Journal of Biological Chemistry 09/2003; 278(32):30317-27. · 4.77 Impact Factor
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Gazi S. Hossain, Johannes V. van Thienen,
Geoff H. Werstuck,
Ji Zhou,
Sudesh K. Sood,
Jeffrey G. Dickhout,
A. B. Lawrence de Koning,
Damu Tang,
Dongcheng Wu,
Erling Falk,
Ranjana Poddar,
Donald W. Jacobsen,
Kezhong Zhang,
Randal J. Kaufman,
Richard C. Austin
[show abstract]
[hide abstract]
ABSTRACT: Hyperhomocysteinemia is an independent risk factor for cardiovascular
disease and accelerates atherosclerosis in
apoE–/– mice. Despite the
observations that homocysteine causes endoplasmic reticulum (ER) stress and
programmed cell death (PCD) in cultured human vascular endothelial cells, the
cellular factors responsible for this effect and their relevance to
atherogenesis have not been completely elucidated. We report here that
homocysteine induces the expression of T-cell death-associated gene 51
(TDAG51), a member of the pleckstrin homology-related domain family, in
cultured human vascular endothelial cells. This effect was observed for other
ER stress-inducing agents, including dithiothreitol and tunicamycin. TDAG51
expression was attenuated in homozygous A/A mutant eukaryotic translation
initiation factor 2α mouse embryonic fibroblasts treated with
homocysteine or tunicamycin, suggesting that ER stress-induced phosphorylation
of eukaryotic translation initiation factor 2α is required for TDAG51
transcriptional activation. Transient overexpression of TDAG51 elicited
significant changes in cell morphology, decreased cell adhesion, and promoted
detachment-mediated PCD. In support of these in vitro findings,
TDAG51 expression was increased and correlated with PCD in the atherosclerotic
lesions from apoE–/– mice fed
hyperhomocysteinemic diets, compared with mice fed a control diet.
Collectively, these findings provide evidence that TDAG51 is induced by
homocysteine, promotes detachment-mediated PCD, and contributes to the
development of atherosclerosis observed in hyperhomocysteinemia.
Journal of Biological Chemistry 08/2003; 278(32):30317-30327. · 4.77 Impact Factor
