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Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells
Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells Tobias Becher, Torsten J Schulze, Melanie Schmitt, Frederik Trinkmann, Ibrahim El-Battrawy, Ibrahim Akin, Thorsten K¨alsch, Martin Borggrefe, Ksenija Stach PII: DOI: Reference:
S0167-5273(16)32641-9 doi:10.1016/j.ijcard.2016.09.122 IJCA 23729
To appear in:
International Journal of Cardiology
Received date: Revised date: Accepted date:
29 May 2016 28 September 2016 29 September 2016
Please cite this article as: Becher Tobias, Schulze Torsten J, Schmitt Melanie, Trinkmann Frederik, El-Battrawy Ibrahim, Akin Ibrahim, K¨alsch Thorsten, Borggrefe Martin, Stach Ksenija, Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells, International Journal of Cardiology (2016), doi:10.1016/j.ijcard.2016.09.122
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ACCEPTED MANUSCRIPT Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells
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Tobias Becher M.D.*1, Torsten J Schulze M.D.*2, Melanie Schmitt M.D.1, Frederik
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Trinkmann M.D.1, Ibrahim El-Battrawy M.D.1, Ibrahim Akin M.D. PhD.1, Thorsten
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Kälsch M.D. PhD.3, Martin Borggrefe M.D. PhD.1, Ksenija Stach M.D.1
From the 1st Department of Medicine, University Medical Centre Mannheim, Medical
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Faculty Mannheim, University of Heidelberg, Germany
Institute of Transfusion Medicine and Immunology, German Red Cross Blood
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Service Baden-Württemberg-Hessen, Medical Faculty Mannheim, University of Heidelberg, Germany 3
Herzzentrum Weinheim, University Medical Centre Mannheim, Medical Faculty
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Mannheim, University of Heidelberg, Germany
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(* these authors contributed equally to this work)
Correspondence to:
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Ksenija Stach, MD.
1st Department of Medicine University Medical Centre Mannheim Medical Faculty Mannheim, University of Heidelberg Theodor-Kutzer-Ufer 1-3 68167 Mannheim, Germany Tel. /Fax: 0049-6213832204 / 0049-6213833821 Email: [email protected]
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Abstract
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Purpose Lipid lowering therapy constitutes the basis of cardiovascular disease
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therapy. The purpose of this study was to investigate effects of ezetimibe, a selective
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inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model.
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Methods After a 24 hour incubation period with ezetimibe (concentration 1, 50, 100 and 1000 ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for
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one hour with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on
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platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on
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endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1.
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Results The increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was
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significantly reduced through pre-incubation with 100 ng/ml and 1000 ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. Conclusion In this in vitro analyses ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis. Key Words Platelets; endothelial cells; ezetimibe; inflammation; atherosclerosis 2
ACCEPTED MANUSCRIPT Introduction
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Ahead of these risk factors chronic inflammation plays an important role in the
through
CD
40
receptors
lead
to
an
increased
expression
of
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platelets
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development of atherosclerotic lesions. Interaction between endothelial cells and
proinflammatory factors [1, 2] by expression of vascular cell adhesion molecule VCAM-1 and intercellular adhesion molecule ICAM-1, resulting in initiate the
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recruitment of neutrophils, monocytes and lymphocytes [3-5]. The extravasation of
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monocytes and their metamorphosis to macrophages mediate the process of plaque formation and atherosclerosis [6].
Hypercholesterolemia induces endothelial
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dysfunction by lipid accumulation in the vascular wall und leading to monocyte-
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derived macrophage-foam cells production. Adhesion of activated platelets to endothelial cells results in secretion of matrix degrading enzymes, the matrix
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metalloproteinases (MMP-1, MMP-2 and MMP-9). MMPs degrade various proteins of the extracellular matrix and promote inflammation and the destruction of the inflamed
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tissue [7].
Cardiovascular risk factors such as hypertension, diabetes, smoking, family history and elevated serum lipid levels contribute to the initiation and progression of atherosclerosis [8]. Lipid lowering therapy constitutes the basis of cardiovascular disease therapy for primary as well as secondary prevention. Intensive lipid lowering therapy with ezetimibe as monotherapy or in combination with simvastatin leads to a reduction of levels of low-density lipoprotein cholesterol (LDL-C) [9, 10]. Ezetimibe is the first example of a new class of lipid lowering drugs that inhibits the absorption of biliary and dietary cholesterol from the small intestine. Besides the effect on absorption of cholesterol in the intestine, ezetimibe has also been shown to affect the metabolism of lipoproteins [11]. It is estimated that ezetimibe lowers diet cholesterol 3
ACCEPTED MANUSCRIPT absorption in the intestine by over 50% [12]. However evidence suggests that statins have additional pleiotropic effects beyond their lipid lowering capacities that may
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partially explain their role in decreasing cardiovascular morbidity and mortality;
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whereas to date there is no clear evidence for pleiotropic effect of ezetimibe.
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Therefore we evaluated in the present study the effect of ezetimibe on platelets and
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endothelial cells in an established in vitro cell model.
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ACCEPTED MANUSCRIPT Methods:
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Coincubation of platelets with ezetimibe pre-incubated endothelial cells
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The umbilical vein was cannulated and perfused with PBS to remove all traces of
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blood; the vein lumen was filled with dispase. After 5-minute incubation at 37 °C, the contents of the vein were gently flushed out with endothelial cell basal medium and collected in sterile tubes. After centrifugation the pellet was resuspended in culture
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medium. Human endothelial cells, obtained by collagenase treatment of term
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umbilical cord veins, were cultured in endothelial cell basal medium (PromoCell) [13] containing 2% fetal calf serum (FCS); 1 μg/ml hydrocortison (HC-500); 0.4%
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endothelial cell growth supplement (ECGS/H-2); 0.1 ng/ml epidermal growth factor
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(hEGF-0.05); 1ng/ml basic fibroblast growth factor (hbFGF-0.5); 5ng/ml amphoterecin and 50 µg/ml gentamicin [14-16]. 12-well plates with confluened endothelial cells per
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well were incubated for 24 hours in different concentrations of ezetimibe (1/50/100/1000 ng/ml). Platelets were prepared from blood of healthy probands as
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described [17, 18]. Washed platelets were stimulated with thrombin (0.5 U/ml) and LPS (1000 ng/ml). Pretreated platelets (final concentration 2x10 8/ml) were added to confluent endothelial monolayers with and without ezetimibe (MSD, SHARP & DOHME GMBH Haar, Germany). After 60-minute coincubation under cell culture conditions, all platelets were removed by gentle washing, which was confirmed by light microscopy. After an additional 6 hours of incubation of the endothelial cells, supernatant was aspirated, centrifuged at 2000g and stored at -80°C [19]. Following this incubation, the expressions of activity markers on platelets, as well as that on endothelial cells were measured by flow cytometry.
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ACCEPTED MANUSCRIPT Flow cytometric analysis Flow cytometric analysis of platelets and endothelial cells was performed by gating in
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forward and side scatter. Platelets were gated back for determination of the
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expression of CD40L and CD62P. For the analysis of platelets, 100 μl of each
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sample were stained for 15 min at room temperature with 10 μl aliquots of mouse anti-human CD40L-FITC antibodies (BD Pharmingen, Heidelberg, Germany) and mouse anti-human CD62P-PE antibodies (Beckman-Coulter, Krefeld, Germany).
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Endothelial cells were gated back for determination of the surface expression of
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ICAM-1, VCAM-1, serine protease urokinase-type plasminogen activator receptor uPAR and membrane type 1 matrix metalloproteinase (MT1-MMP). For the analysis of endothelial cells, 100 μl of each sample were stained for 15 min at room
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temperature with 10 μl aliquots of anti-human CD54 PE-Cy5 (ICAM-1 from BD Pharmingen, Heidelberg, Germany), anti-human CD106-FITC (VCAM-1 from R&D
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Systems, Inc., Wiesbaden, Germany), anti-human CD87-FITC (uPAR from American diagnostica inc., Stamford, UK), anti-human MT1-MMP (Ab-1) Mouse mAb (114-IF2)-
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PE (Merck chemicals Ltd., Nottingham, UK). Corresponding isotypes (Beckman Coulter, Marseille, France) were used as a control. All flow cytometric analysis was performed on an EPICS XL-MCL analyzer (Beckman Coulter, Krefeld, Germany) equipped with an argon laser tuned at 488 nm. Compensation of the four channel fluorescence was precisely adjusted using Cyto-CompTM reagents and Cyto-TrolTM control cells (Coulter Immunotech / Krefeld, Germany).
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ACCEPTED MANUSCRIPT Enzyme linked immunosorbent assay (ELISA) Concentrations of MMP-1 (Human, Biotrak ELISA System GE Healthcare, UK
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Limited), Interleukin-6 (IL-6) (Human IL-6 Immunoassay R&D Systems, Inc.,
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Wiesbaden, Germany) and monocyte chemotactic protein-1 (MCP-1) (Human MCP-
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1/CCL2 Immunoassay R&D Systems, Inc., Wiesbaden, Germany) in supernatants were determined by sandwich-type immunoassay according to the manufacturer instructions. All concentration analysis was performed on an ELISA-Reader - Lab
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Systems Multiskan RC (Lab systems, Finland). Genesis Lite Software, ELISA
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Multiskan RC was used for data acquisition and evaluation.
Statistical Analysis:
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All calculations were performed using SAS release 9.2 (SAS institute inc. Cary NC USA). Numerical data were expressed as means ± SD. The Wilcoxon-test was
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significant.
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applied as non-parametric test. A two-tailed probability value <0.05 was considered
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ACCEPTED MANUSCRIPT Results:
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Effects of pre-incubation with ezetimibe on endothelial cell surface markers:
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HUVEC pre-incubation with ezetimibe resulted in decreased surface expression of
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uPAR under stimulation with LPS stimulated platelets and by direct endothelial contact with resting platelets as compared to HUVEC without ezetimibe. Pre-
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incubation with 100 ng/ml as well as 1000 ng/ml ezetimibe significantly reduced surface expression of uPAR from 0.90±0.53 to 0.44±0.18 (p=0.041) and from
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0.90±0.53 to 0.34±0.10 (p=0.009) after contact with LPS stimulated platelets (figure
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Endothelial cell mediated and direct effects of ezetimibe on platelets:
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Platelets in direct contact with ezetimibe pre-incubated endothelial cells showed a significant reduction in their CD62P expression (figure 2, table 1).
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However platelets in direct contact with ezetimibe pre-incubated endothelial cells showed no significant reduction in their CD40L expression. When compared to platelets incubated with untreated endothelial cells. To discriminate between effects mediated by pre-treated endothelial cells and possible direct effects of ezetimibe on platelets, platelets were directly incubated with ezetimibe alone for one hour. Hereby incubation with ezetimibe significantly decreased CD62P expression on thrombin stimulated platelets and on thrombin and LPS stimulated platelets (figure 3, table 2). CD40L expression on resting platelets after direct incubation with 1 ng/ml ezetimibe was significantly reduced from 14.521.47 to 12.251.07 (p=0.015) and on thrombin 8
ACCEPTED MANUSCRIPT stimulated platelets from 36.252.69 to 27.704.94 (p=0.015). However, after direct incubation with 50 ng/ml as well as 100 ng/ml ezetimibe CD 40L expression was
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significantly reduced by stimulation with thrombin (figure 4).
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Effects of pre-incubation with ezetimibe on cytokine production: Supernatant concentrations of IL-6 upon HUVEC stimulation with resting platelets
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were significantly reduced by HUVEC pre-incubation with 1/50/1000 ng/ml ezetimibe (figure 5). Supernatant concentrations of MMP-1 upon HUVEC stimulation with LPS
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activated platelets were significantly reduced by HUVEC pre-incubation with 50 ng/ml ezetimibe (figure 6).
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Ezetimibe had no significant effect on supernatant concentrations of MCP-1 upon
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HUVEC stimulation with activated platelets.
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ACCEPTED MANUSCRIPT Discussion Atherosclerosis is considered a chronic inflammatory disease with activated platelets,
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endothelial cells and locally released cytokines playing a prominent role [20] and is
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associated with endothelial dysfunction and other profound changes in the vascular
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system [21]. It is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial cells, smooth muscle cells, platelets, extracellular
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matrix and a lipid-rich core. The role of dyslipidaemia as a risk factor for cardiovascular disease is well demonstrated [22].
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Lipid lowering medication therapy is one of the cornerstones of cardiovascular disease therapy. Medical treatment with ezetimibe being the first lipid lowering drug
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inhibiting the absorption of cholesterol from the small intestine by blocking NPC1L1
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protein [23] results in a significant reduction of total cholesterol and LDL cholesterol levels [24].
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In a previous study, we were able to demonstrate that platelets in direct contact with statins but without endothelial cell contact show a significant reduction in the CD40L
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expression [25]. The increased expression of VCAM-1 and uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with simvastatin or atorvastatin [25]. In the current study, after pre-incubation with ezetimibe, the expression of uPAR on endothelial cells was significantly decreased under stimulation with LPS activated platelets. With increasing concentration of ezetimibe the expression of uPAR on endothelial cells after incubation with LPS stimulated platelets was decreased. The serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in forming the cap of the plaque. uPAR is a part of plasminogen activation system and facilitates monocyte-endothelial cells interaction. In a comparable study from Sager at al., adding ezetimibe to 10
ACCEPTED MANUSCRIPT simvastatin resulted in a two-fold greater reduction in CRP concentration compared to treatment with simvastatin alone [26]. Both CRP concentration and uPAR support
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the development of atherosclerosis and are decreased by ezetimibe. Davis et al.
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atherogenesis in apolipoprotein E knockout mice [27].
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demonstrated that ezetimibe reduced plasma cholesterol levels and inhibited
However, so far, only little evidence exists regarding pleiotropic effects of ezetimibe. In this study ezetimibe significantly reduced the expression of established
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proatherogenic activity and progression markers on platelets and endothelial cells in
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an in vitro cell model. The expression of CD62P and CD40L on platelets stimulated with thrombin was significantly decreased after direct contact with ezetimibe for one hour under proinflammatory conditions. These results suggest possible similar effects
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of ezetimibe on platelets and endothelial cells under proinflammatory conditions in comparison to statins. The observed direct effects of ezetimibe on CD62P and
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CD40L on platelets may be of relevance, since platelets are in periodical contact with vascular endothelial cells and atherosclerotic processes partially are located in the
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blood vessel wall.
Previous studies described that ezetimibe reduced VCAM-1 expression in ApoEdeficient mice and in both ApoE- and eNOS-deficient mice. This effect seems to be independently of eNOS function [28]. Although the anti-inflammatory signalling mechanisms of ezetimibe are still unknown, perhaps ezetimibe interacts with the aminopeptidase N/CD13 (APN/CD13) [29], a transmembrane protein implicated in adhesion and cell-cell interaction [30]. Orsó et al. reported that ezetimibe decreased the surface expression of APN/CD13 and of CD16, CD64 and the scavenger receptor CD36 in monocyte/macrophages [31]. Gómez-Garre et al. demonstrated decreased macrophage content and monocyte chemoattractant protein-1 expression in atherosclerotic lesions pre-treated with ezetimibe. Furthermore, ezetimibe reduced 11
ACCEPTED MANUSCRIPT the increased activity of nuclear factor κB in peripheral blood leucocytes in rabbits with atherosclerosis [32].
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However, in contrast to these in vitro evaluations, in vivo studies demonstrated
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different results regarding the effects of ezetimibe. In the ENHANCE trial, the
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comparison of 80 mg simvastatin plus either placebo or 10 mg ezetimibe revealed no significant differences between both groups [33]. Recently, the 7 year results of
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IMPROVE-IT showed significant reduction of myocardial infarction and nonfatal stroke through the additional therapy with ezetimibe without any impact on as well as
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cardiovascular mortality rate. Additional findings by Cannon at al. showed reduction of cardiovascular events in stable patients who had an acute coronary syndrome and
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who had LDL cholesterol levels within guideline recommendations after combination
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simvastatin with ezetimibe [34].
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Considering the discrepant results of in vivo and in vitro trials there is a need for more dedicated trials addressing the in vivo and in vitro pathophysiological effects of ezetimibe on atherosclerotic burden not only explained by reduction of cholesterol
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resorption.
In conclusion the present study revealed significant in vitro effects of ezetimibe on endothelial cells and platelets providing further evidence of possible pleiotropic therapeutic relevance of ezetimibe in addition to its inhibition of intestinal cholesterol absorption.
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Acknowledgements: work
was
supported
by
MSD,
SHARP
&
DOHME
GMBH
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This
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Lindenplatz 1, 85540 Haar, Germany. The data has never been presented in the
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scientific meeting. Nobody else was involved who do not meet the criteria of authorship. Authors have the permission to acknowledge from all those mentioned in
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the above section.
Fundings:
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All sources of funding are declared in the Acknowledgements. All authors certify that all financial and material support for the conduct of these studies clearly described in
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the Acknowledgements section of the manuscript. This research received no grant
Disclosures:
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cost of supplies.
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from any funding agency in the public, commercial or not-for-profit sectors, only for
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The Authors declare that there is no conflict of interest.
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ACCEPTED MANUSCRIPT Figure Legends: Figure 1: Surface expression of uPAR on HUVEC with and without 24 h pre-incubation with 1/50/100/1000 ng/ml ezetimibe.
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N=6; PLT = platelets; LPS = lipopolysaccharide
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(* p < 0.05 vs. non pre-treated endothelial cells)
Figure 2: Surface expression of CD62P on stimulated platelets with thrombin (A) or with thrombin and lipopolysaccharide (B) after 1h adherence to HUVEC with and without ezetimibe pre-treatment.
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(* p<0.05 vs. non stimulated platelets)
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N=6; PLT = platelets; Thr. = thrombin; LPS = lipopolysaccharide
Figure 3: Surface expression of CD62P on stimulated platelets with thrombin (A) or with thrombin and lipopolysaccharide (B) without endothelial contact.
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N=6; PLT = platelets; Thr. = thrombin; LPS = lipopolysaccharide
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(* p < 0.05 vs. non pre-treated platelets)
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Figure 4: Surface expression of CD40L on platelets without endothelial contact. N=6; PLT = platelets; Thr. = thrombin; LPS = lipopolysaccharide
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(* p < 0.05 vs. non pre-treated platelets)
Figure 5: Supernatant concentrations of soluble IL-6 upon stimulation with resting platelets on HUVEC with and without pre-incubation with ezetimibe. (* p < 0.05 vs. non pre-treated endothelial cells)
Figure 6: Supernatant concentrations of soluble MMP-1 upon stimulation with LPS activated platelets on HUVEC with and without pre-incubation with ezetimibe. (* p < 0.05 vs. non pre-treated endothelial cells)
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ACCEPTED MANUSCRIPT Tables: Table 1 without
1 ng/ml
50 ng/ml
100 ng/ml
PLT
3.2 ± 2.9
2.3 ± 1.1
2.1 ± 0.9
2.1 ± 1.0
1.9 ± 0.8
PLT + Thr.
11.5 ± 4.3
8.6 ± 1.7
9.2 ± 2.6
7.4 ± 1.8
6.4 ± 1.5
p=0.026
p=0.009
7.6 ± 2.1
6.4 ± 1.7
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7.3 ± 1.8
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9.5 ± 3.0
1000 ng/ml
p=0.015
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PLT + Thr. + LPS 10.5 ± 4.7
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Ezetimibe
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ACCEPTED MANUSCRIPT Table2 without
1 ng/ml
50 ng/ml
100 ng/ml
1000 ng/ml
PLT
1.8 ± 0.2
1.8 ± 0.4
1.5 ± 0.2
1.5 ± 0.2
1.4 ± 0.1
p=0.026
p=0.041
p=0.015
19.5 ± 0.6
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13.7 ± 3.0
14.5 ± 2.7
14.3 ± 1.7
p=0.002
p=0.002
p=0.002
16.4 ± 1.4
12.7 ± 2.7
12.5 ± 3.4
13.4 ± 3.7
p=0.002
p=0.002
p=0.002
p=0.002
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PLT + Thr. + LPS
15.6 ± 1.2
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18.9 ± 0.7
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PLT + Thr.
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Ezetimibe
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S0167-5273(16)32641-9 doi:10.1016/j.ijcard.2016.09.122 IJCA 23729
To appear in:
International Journal of Cardiology
Received date: Revised date: Accepted date:
29 May 2016 28 September 2016 29 September 2016
Please cite this article as: Becher Tobias, Schulze Torsten J, Schmitt Melanie, Trinkmann Frederik, El-Battrawy Ibrahim, Akin Ibrahim, K¨alsch Thorsten, Borggrefe Martin, Stach Ksenija, Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells, International Journal of Cardiology (2016), doi:10.1016/j.ijcard.2016.09.122
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ACCEPTED MANUSCRIPT Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells
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Tobias Becher M.D.*1, Torsten J Schulze M.D.*2, Melanie Schmitt M.D.1, Frederik
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Trinkmann M.D.1, Ibrahim El-Battrawy M.D.1, Ibrahim Akin M.D. PhD.1, Thorsten
1
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Kälsch M.D. PhD.3, Martin Borggrefe M.D. PhD.1, Ksenija Stach M.D.1
From the 1st Department of Medicine, University Medical Centre Mannheim, Medical
2
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Faculty Mannheim, University of Heidelberg, Germany
Institute of Transfusion Medicine and Immunology, German Red Cross Blood
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Service Baden-Württemberg-Hessen, Medical Faculty Mannheim, University of Heidelberg, Germany 3
Herzzentrum Weinheim, University Medical Centre Mannheim, Medical Faculty
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Mannheim, University of Heidelberg, Germany
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(* these authors contributed equally to this work)
Correspondence to:
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Ksenija Stach, MD.
1st Department of Medicine University Medical Centre Mannheim Medical Faculty Mannheim, University of Heidelberg Theodor-Kutzer-Ufer 1-3 68167 Mannheim, Germany Tel. /Fax: 0049-6213832204 / 0049-6213833821 Email: [email protected]
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Abstract
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Purpose Lipid lowering therapy constitutes the basis of cardiovascular disease
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therapy. The purpose of this study was to investigate effects of ezetimibe, a selective
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inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model.
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Methods After a 24 hour incubation period with ezetimibe (concentration 1, 50, 100 and 1000 ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for
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one hour with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on
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platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on
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endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1.
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Results The increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was
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significantly reduced through pre-incubation with 100 ng/ml and 1000 ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. Conclusion In this in vitro analyses ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis. Key Words Platelets; endothelial cells; ezetimibe; inflammation; atherosclerosis 2
ACCEPTED MANUSCRIPT Introduction
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Ahead of these risk factors chronic inflammation plays an important role in the
through
CD
40
receptors
lead
to
an
increased
expression
of
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platelets
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development of atherosclerotic lesions. Interaction between endothelial cells and
proinflammatory factors [1, 2] by expression of vascular cell adhesion molecule VCAM-1 and intercellular adhesion molecule ICAM-1, resulting in initiate the
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recruitment of neutrophils, monocytes and lymphocytes [3-5]. The extravasation of
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monocytes and their metamorphosis to macrophages mediate the process of plaque formation and atherosclerosis [6].
Hypercholesterolemia induces endothelial
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dysfunction by lipid accumulation in the vascular wall und leading to monocyte-
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derived macrophage-foam cells production. Adhesion of activated platelets to endothelial cells results in secretion of matrix degrading enzymes, the matrix
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metalloproteinases (MMP-1, MMP-2 and MMP-9). MMPs degrade various proteins of the extracellular matrix and promote inflammation and the destruction of the inflamed
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tissue [7].
Cardiovascular risk factors such as hypertension, diabetes, smoking, family history and elevated serum lipid levels contribute to the initiation and progression of atherosclerosis [8]. Lipid lowering therapy constitutes the basis of cardiovascular disease therapy for primary as well as secondary prevention. Intensive lipid lowering therapy with ezetimibe as monotherapy or in combination with simvastatin leads to a reduction of levels of low-density lipoprotein cholesterol (LDL-C) [9, 10]. Ezetimibe is the first example of a new class of lipid lowering drugs that inhibits the absorption of biliary and dietary cholesterol from the small intestine. Besides the effect on absorption of cholesterol in the intestine, ezetimibe has also been shown to affect the metabolism of lipoproteins [11]. It is estimated that ezetimibe lowers diet cholesterol 3
ACCEPTED MANUSCRIPT absorption in the intestine by over 50% [12]. However evidence suggests that statins have additional pleiotropic effects beyond their lipid lowering capacities that may
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partially explain their role in decreasing cardiovascular morbidity and mortality;
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whereas to date there is no clear evidence for pleiotropic effect of ezetimibe.
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Therefore we evaluated in the present study the effect of ezetimibe on platelets and
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endothelial cells in an established in vitro cell model.
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ACCEPTED MANUSCRIPT Methods:
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Coincubation of platelets with ezetimibe pre-incubated endothelial cells
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The umbilical vein was cannulated and perfused with PBS to remove all traces of
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blood; the vein lumen was filled with dispase. After 5-minute incubation at 37 °C, the contents of the vein were gently flushed out with endothelial cell basal medium and collected in sterile tubes. After centrifugation the pellet was resuspended in culture
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medium. Human endothelial cells, obtained by collagenase treatment of term
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umbilical cord veins, were cultured in endothelial cell basal medium (PromoCell) [13] containing 2% fetal calf serum (FCS); 1 μg/ml hydrocortison (HC-500); 0.4%
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endothelial cell growth supplement (ECGS/H-2); 0.1 ng/ml epidermal growth factor
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(hEGF-0.05); 1ng/ml basic fibroblast growth factor (hbFGF-0.5); 5ng/ml amphoterecin and 50 µg/ml gentamicin [14-16]. 12-well plates with confluened endothelial cells per
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well were incubated for 24 hours in different concentrations of ezetimibe (1/50/100/1000 ng/ml). Platelets were prepared from blood of healthy probands as
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described [17, 18]. Washed platelets were stimulated with thrombin (0.5 U/ml) and LPS (1000 ng/ml). Pretreated platelets (final concentration 2x10 8/ml) were added to confluent endothelial monolayers with and without ezetimibe (MSD, SHARP & DOHME GMBH Haar, Germany). After 60-minute coincubation under cell culture conditions, all platelets were removed by gentle washing, which was confirmed by light microscopy. After an additional 6 hours of incubation of the endothelial cells, supernatant was aspirated, centrifuged at 2000g and stored at -80°C [19]. Following this incubation, the expressions of activity markers on platelets, as well as that on endothelial cells were measured by flow cytometry.
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ACCEPTED MANUSCRIPT Flow cytometric analysis Flow cytometric analysis of platelets and endothelial cells was performed by gating in
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forward and side scatter. Platelets were gated back for determination of the
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expression of CD40L and CD62P. For the analysis of platelets, 100 μl of each
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sample were stained for 15 min at room temperature with 10 μl aliquots of mouse anti-human CD40L-FITC antibodies (BD Pharmingen, Heidelberg, Germany) and mouse anti-human CD62P-PE antibodies (Beckman-Coulter, Krefeld, Germany).
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Endothelial cells were gated back for determination of the surface expression of
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ICAM-1, VCAM-1, serine protease urokinase-type plasminogen activator receptor uPAR and membrane type 1 matrix metalloproteinase (MT1-MMP). For the analysis of endothelial cells, 100 μl of each sample were stained for 15 min at room
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temperature with 10 μl aliquots of anti-human CD54 PE-Cy5 (ICAM-1 from BD Pharmingen, Heidelberg, Germany), anti-human CD106-FITC (VCAM-1 from R&D
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Systems, Inc., Wiesbaden, Germany), anti-human CD87-FITC (uPAR from American diagnostica inc., Stamford, UK), anti-human MT1-MMP (Ab-1) Mouse mAb (114-IF2)-
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PE (Merck chemicals Ltd., Nottingham, UK). Corresponding isotypes (Beckman Coulter, Marseille, France) were used as a control. All flow cytometric analysis was performed on an EPICS XL-MCL analyzer (Beckman Coulter, Krefeld, Germany) equipped with an argon laser tuned at 488 nm. Compensation of the four channel fluorescence was precisely adjusted using Cyto-CompTM reagents and Cyto-TrolTM control cells (Coulter Immunotech / Krefeld, Germany).
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ACCEPTED MANUSCRIPT Enzyme linked immunosorbent assay (ELISA) Concentrations of MMP-1 (Human, Biotrak ELISA System GE Healthcare, UK
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Limited), Interleukin-6 (IL-6) (Human IL-6 Immunoassay R&D Systems, Inc.,
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Wiesbaden, Germany) and monocyte chemotactic protein-1 (MCP-1) (Human MCP-
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1/CCL2 Immunoassay R&D Systems, Inc., Wiesbaden, Germany) in supernatants were determined by sandwich-type immunoassay according to the manufacturer instructions. All concentration analysis was performed on an ELISA-Reader - Lab
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Systems Multiskan RC (Lab systems, Finland). Genesis Lite Software, ELISA
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Multiskan RC was used for data acquisition and evaluation.
Statistical Analysis:
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All calculations were performed using SAS release 9.2 (SAS institute inc. Cary NC USA). Numerical data were expressed as means ± SD. The Wilcoxon-test was
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significant.
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applied as non-parametric test. A two-tailed probability value <0.05 was considered
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ACCEPTED MANUSCRIPT Results:
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Effects of pre-incubation with ezetimibe on endothelial cell surface markers:
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HUVEC pre-incubation with ezetimibe resulted in decreased surface expression of
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uPAR under stimulation with LPS stimulated platelets and by direct endothelial contact with resting platelets as compared to HUVEC without ezetimibe. Pre-
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incubation with 100 ng/ml as well as 1000 ng/ml ezetimibe significantly reduced surface expression of uPAR from 0.90±0.53 to 0.44±0.18 (p=0.041) and from
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0.90±0.53 to 0.34±0.10 (p=0.009) after contact with LPS stimulated platelets (figure
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1).
Endothelial cell mediated and direct effects of ezetimibe on platelets:
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Platelets in direct contact with ezetimibe pre-incubated endothelial cells showed a significant reduction in their CD62P expression (figure 2, table 1).
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However platelets in direct contact with ezetimibe pre-incubated endothelial cells showed no significant reduction in their CD40L expression. When compared to platelets incubated with untreated endothelial cells. To discriminate between effects mediated by pre-treated endothelial cells and possible direct effects of ezetimibe on platelets, platelets were directly incubated with ezetimibe alone for one hour. Hereby incubation with ezetimibe significantly decreased CD62P expression on thrombin stimulated platelets and on thrombin and LPS stimulated platelets (figure 3, table 2). CD40L expression on resting platelets after direct incubation with 1 ng/ml ezetimibe was significantly reduced from 14.521.47 to 12.251.07 (p=0.015) and on thrombin 8
ACCEPTED MANUSCRIPT stimulated platelets from 36.252.69 to 27.704.94 (p=0.015). However, after direct incubation with 50 ng/ml as well as 100 ng/ml ezetimibe CD 40L expression was
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significantly reduced by stimulation with thrombin (figure 4).
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Effects of pre-incubation with ezetimibe on cytokine production: Supernatant concentrations of IL-6 upon HUVEC stimulation with resting platelets
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were significantly reduced by HUVEC pre-incubation with 1/50/1000 ng/ml ezetimibe (figure 5). Supernatant concentrations of MMP-1 upon HUVEC stimulation with LPS
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activated platelets were significantly reduced by HUVEC pre-incubation with 50 ng/ml ezetimibe (figure 6).
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Ezetimibe had no significant effect on supernatant concentrations of MCP-1 upon
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HUVEC stimulation with activated platelets.
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ACCEPTED MANUSCRIPT Discussion Atherosclerosis is considered a chronic inflammatory disease with activated platelets,
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endothelial cells and locally released cytokines playing a prominent role [20] and is
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associated with endothelial dysfunction and other profound changes in the vascular
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system [21]. It is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial cells, smooth muscle cells, platelets, extracellular
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matrix and a lipid-rich core. The role of dyslipidaemia as a risk factor for cardiovascular disease is well demonstrated [22].
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Lipid lowering medication therapy is one of the cornerstones of cardiovascular disease therapy. Medical treatment with ezetimibe being the first lipid lowering drug
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inhibiting the absorption of cholesterol from the small intestine by blocking NPC1L1
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protein [23] results in a significant reduction of total cholesterol and LDL cholesterol levels [24].
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In a previous study, we were able to demonstrate that platelets in direct contact with statins but without endothelial cell contact show a significant reduction in the CD40L
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expression [25]. The increased expression of VCAM-1 and uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with simvastatin or atorvastatin [25]. In the current study, after pre-incubation with ezetimibe, the expression of uPAR on endothelial cells was significantly decreased under stimulation with LPS activated platelets. With increasing concentration of ezetimibe the expression of uPAR on endothelial cells after incubation with LPS stimulated platelets was decreased. The serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in forming the cap of the plaque. uPAR is a part of plasminogen activation system and facilitates monocyte-endothelial cells interaction. In a comparable study from Sager at al., adding ezetimibe to 10
ACCEPTED MANUSCRIPT simvastatin resulted in a two-fold greater reduction in CRP concentration compared to treatment with simvastatin alone [26]. Both CRP concentration and uPAR support
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the development of atherosclerosis and are decreased by ezetimibe. Davis et al.
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atherogenesis in apolipoprotein E knockout mice [27].
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demonstrated that ezetimibe reduced plasma cholesterol levels and inhibited
However, so far, only little evidence exists regarding pleiotropic effects of ezetimibe. In this study ezetimibe significantly reduced the expression of established
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proatherogenic activity and progression markers on platelets and endothelial cells in
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an in vitro cell model. The expression of CD62P and CD40L on platelets stimulated with thrombin was significantly decreased after direct contact with ezetimibe for one hour under proinflammatory conditions. These results suggest possible similar effects
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of ezetimibe on platelets and endothelial cells under proinflammatory conditions in comparison to statins. The observed direct effects of ezetimibe on CD62P and
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CD40L on platelets may be of relevance, since platelets are in periodical contact with vascular endothelial cells and atherosclerotic processes partially are located in the
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blood vessel wall.
Previous studies described that ezetimibe reduced VCAM-1 expression in ApoEdeficient mice and in both ApoE- and eNOS-deficient mice. This effect seems to be independently of eNOS function [28]. Although the anti-inflammatory signalling mechanisms of ezetimibe are still unknown, perhaps ezetimibe interacts with the aminopeptidase N/CD13 (APN/CD13) [29], a transmembrane protein implicated in adhesion and cell-cell interaction [30]. Orsó et al. reported that ezetimibe decreased the surface expression of APN/CD13 and of CD16, CD64 and the scavenger receptor CD36 in monocyte/macrophages [31]. Gómez-Garre et al. demonstrated decreased macrophage content and monocyte chemoattractant protein-1 expression in atherosclerotic lesions pre-treated with ezetimibe. Furthermore, ezetimibe reduced 11
ACCEPTED MANUSCRIPT the increased activity of nuclear factor κB in peripheral blood leucocytes in rabbits with atherosclerosis [32].
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However, in contrast to these in vitro evaluations, in vivo studies demonstrated
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different results regarding the effects of ezetimibe. In the ENHANCE trial, the
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comparison of 80 mg simvastatin plus either placebo or 10 mg ezetimibe revealed no significant differences between both groups [33]. Recently, the 7 year results of
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IMPROVE-IT showed significant reduction of myocardial infarction and nonfatal stroke through the additional therapy with ezetimibe without any impact on as well as
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cardiovascular mortality rate. Additional findings by Cannon at al. showed reduction of cardiovascular events in stable patients who had an acute coronary syndrome and
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who had LDL cholesterol levels within guideline recommendations after combination
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simvastatin with ezetimibe [34].
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Considering the discrepant results of in vivo and in vitro trials there is a need for more dedicated trials addressing the in vivo and in vitro pathophysiological effects of ezetimibe on atherosclerotic burden not only explained by reduction of cholesterol
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resorption.
In conclusion the present study revealed significant in vitro effects of ezetimibe on endothelial cells and platelets providing further evidence of possible pleiotropic therapeutic relevance of ezetimibe in addition to its inhibition of intestinal cholesterol absorption.
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Acknowledgements: work
was
supported
by
MSD,
SHARP
&
DOHME
GMBH
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This
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Lindenplatz 1, 85540 Haar, Germany. The data has never been presented in the
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scientific meeting. Nobody else was involved who do not meet the criteria of authorship. Authors have the permission to acknowledge from all those mentioned in
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the above section.
Fundings:
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All sources of funding are declared in the Acknowledgements. All authors certify that all financial and material support for the conduct of these studies clearly described in
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the Acknowledgements section of the manuscript. This research received no grant
Disclosures:
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cost of supplies.
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from any funding agency in the public, commercial or not-for-profit sectors, only for
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The Authors declare that there is no conflict of interest.
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ACCEPTED MANUSCRIPT Figure Legends: Figure 1: Surface expression of uPAR on HUVEC with and without 24 h pre-incubation with 1/50/100/1000 ng/ml ezetimibe.
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N=6; PLT = platelets; LPS = lipopolysaccharide
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(* p < 0.05 vs. non pre-treated endothelial cells)
Figure 2: Surface expression of CD62P on stimulated platelets with thrombin (A) or with thrombin and lipopolysaccharide (B) after 1h adherence to HUVEC with and without ezetimibe pre-treatment.
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(* p<0.05 vs. non stimulated platelets)
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N=6; PLT = platelets; Thr. = thrombin; LPS = lipopolysaccharide
Figure 3: Surface expression of CD62P on stimulated platelets with thrombin (A) or with thrombin and lipopolysaccharide (B) without endothelial contact.
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N=6; PLT = platelets; Thr. = thrombin; LPS = lipopolysaccharide
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(* p < 0.05 vs. non pre-treated platelets)
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Figure 4: Surface expression of CD40L on platelets without endothelial contact. N=6; PLT = platelets; Thr. = thrombin; LPS = lipopolysaccharide
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(* p < 0.05 vs. non pre-treated platelets)
Figure 5: Supernatant concentrations of soluble IL-6 upon stimulation with resting platelets on HUVEC with and without pre-incubation with ezetimibe. (* p < 0.05 vs. non pre-treated endothelial cells)
Figure 6: Supernatant concentrations of soluble MMP-1 upon stimulation with LPS activated platelets on HUVEC with and without pre-incubation with ezetimibe. (* p < 0.05 vs. non pre-treated endothelial cells)
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ACCEPTED MANUSCRIPT Tables: Table 1 without
1 ng/ml
50 ng/ml
100 ng/ml
PLT
3.2 ± 2.9
2.3 ± 1.1
2.1 ± 0.9
2.1 ± 1.0
1.9 ± 0.8
PLT + Thr.
11.5 ± 4.3
8.6 ± 1.7
9.2 ± 2.6
7.4 ± 1.8
6.4 ± 1.5
p=0.026
p=0.009
7.6 ± 2.1
6.4 ± 1.7
IP
SC R
7.3 ± 1.8
NU
9.5 ± 3.0
1000 ng/ml
p=0.015
AC
CE P
TE
D
MA
PLT + Thr. + LPS 10.5 ± 4.7
T
Ezetimibe
27
ACCEPTED MANUSCRIPT Table2 without
1 ng/ml
50 ng/ml
100 ng/ml
1000 ng/ml
PLT
1.8 ± 0.2
1.8 ± 0.4
1.5 ± 0.2
1.5 ± 0.2
1.4 ± 0.1
p=0.026
p=0.041
p=0.015
19.5 ± 0.6
IP
13.7 ± 3.0
14.5 ± 2.7
14.3 ± 1.7
p=0.002
p=0.002
p=0.002
16.4 ± 1.4
12.7 ± 2.7
12.5 ± 3.4
13.4 ± 3.7
p=0.002
p=0.002
p=0.002
p=0.002
AC
CE P
TE
D
MA
PLT + Thr. + LPS
15.6 ± 1.2
SC R
18.9 ± 0.7
NU
PLT + Thr.
T
Ezetimibe
28