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Title: Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

Abstract

Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cellmore » proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.« less

Authors:
 [1]; ;  [2];  [1];  [3];  [1]
  1. Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)
  2. NARILIS, URBC, University of Namur, Namur (Belgium)
  3. Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium)
Publication Date:
OSTI Identifier:
22242190
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 441; Journal Issue: 2; Other Information: Copyright (c) 2013 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ARACHIDONIC ACID; ARTERIOSCLEROSIS; CELL PROLIFERATION; CONCENTRATION RATIO; CORONARIES; ENZYMES; GROWTH FACTORS; HYPERTROPHY; INFLAMMATION; INSULIN; MESSENGER-RNA; MUSCLES; POLYMERASE CHAIN REACTION; RECEPTORS; VEINS

Citation Formats

Petri, Marcelo H., Tellier, Céline, Michiels, Carine, Ellertsen, Ingvill, Dogné, Jean-Michel, and Bäck, Magnus, E-mail: Magnus.Back@ki.se. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells. United States: N. p., 2013. Web. doi:10.1016/J.BBRC.2013.10.078.
Petri, Marcelo H., Tellier, Céline, Michiels, Carine, Ellertsen, Ingvill, Dogné, Jean-Michel, & Bäck, Magnus, E-mail: Magnus.Back@ki.se. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells. United States. doi:10.1016/J.BBRC.2013.10.078.
Petri, Marcelo H., Tellier, Céline, Michiels, Carine, Ellertsen, Ingvill, Dogné, Jean-Michel, and Bäck, Magnus, E-mail: Magnus.Back@ki.se. 2013. "Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells". United States. doi:10.1016/J.BBRC.2013.10.078.
@article{osti_22242190,
title = {Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells},
author = {Petri, Marcelo H. and Tellier, Céline and Michiels, Carine and Ellertsen, Ingvill and Dogné, Jean-Michel and Bäck, Magnus, E-mail: Magnus.Back@ki.se},
abstractNote = {Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.},
doi = {10.1016/J.BBRC.2013.10.078},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 441,
place = {United States},
year = 2013,
month =
}
  • The present study utilizes a newly synthesized TXA{sub 2}/PGH{sub 2} mimetic, I-BOP, to characterize the TXA{sub 2}/PGH{sub 2} receptor in suspensions of cultured human vascular smooth muscle cells. ({sup 125}I)-BOP bound in a saturable and specific manner (K{sub d} = 2.6 {plus minus} 0.6 nM; B{sub max} = 33,540 {plus minus} 6,200 sites/cell; 69 fmoles/mg protein, n=12). Competition binding assays were performed with ({sup 125}I)-BOP and the TXA{sub 2}/PGH{sub 2} receptor antagonists SQ29548, L657925 and L657926 and the receptor agonist U46619. I-BOP induced concentration-dependent increases in intracellular free calcium which were inhibited by SQ29548. The results provide radioligand binding evidencemore » for the presence of a TXA{sub 2}/PGH{sub 2} receptor in human vascular smooth muscle cells.« less
  • Thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) are potent vasoconstrictors whose contractile effects are mediated by increases in cellular calcium. Stable analogues of these compounds have shown calcium ionophore activity at high concentrations. To determine if effects of TXA2/PGH2 analogues on /sup 45/Ca/sup 2 +/ fluxes are receptor mediated, the effects of the stable TXA2/PGH2 mimetic U46619 and the TXA2/PGH2 receptor antagonist I-PTA-OH on /sup 45/Ca/+ fluxes in cultured human vascular smooth muscle cells were studied. The smooth muscle cells were cultured from human saphenous vein explants, and they retained the morphologic and immunologic characteristics of vascular smooth muscle cells. U46619 stimulatedmore » /sup 45/Ca/sup 2 +/ efflux in a dose-dependent manner with an EC50 of 398 +/- 26 nM (n = 4). The maximal /sup 45/Ca/sup 2 +/ efflux in response to U46619 (5 microM) was significantly greater (p = 0.006) than the /sup 45/Ca/sup 2 +/ efflux induced by KCl (40 mM). I-PTA-OH inhibited the U46619-induced /sup 45/Ca/sup 2 +/ efflux but had no effect on KCl-induced /sup 45/Ca/sup 2 +/ efflux. These results suggest that the effects of U46619 in increasing vascular smooth muscle cell calcium efflux are receptor mediated. Furthermore, vascular smooth muscle cells with functional TXA2/PGH2 receptors were cultured from human saphenous veins and provide a potentially useful in vitro system for the further study of TXA2/PGH2 receptor-mediated phenomena in human vascular tissue.« less
  • Highlights: • The dedifferentiation of VSMCs in arterial intima is involved in atherosclerosis. • 5-HT showed proliferative effect on VSMCs which was abolished by sarpogrelate. • 5-HT enhanced expression of LR11 mRNA in VSMCs which was abolished by sarpogrelate. • 5-HT suppressed 7KCHO-induced apoptosis of VSMCs via caspase-3/7-dependent pathway. • The mechanisms explain the 5-HT-induced remodeling of arterial structure. - Abstract: Serotonin (5-HT) is a known mitogen for vascular smooth muscle cells (VSMCs). The dedifferentiation and proliferation/apoptosis of VSMCs in the arterial intima represent one of the atherosclerotic changes. LR11, a member of low-density lipoprotein receptor family, may contribute tomore » the proliferation of VSMCs in neointimal hyperplasia. We conducted an in vitro study to investigate whether 5-HT is involved in LR11 expression in human VSMCs and apoptosis of VSMCs induced by 7-ketocholesterol (7KCHO), an oxysterol that destabilizes plaque. 5-HT enhanced the proliferation of VSMCs, and this effect was abolished by sarpogrelate, a selective 5-HT2A receptor antagonist. Sarpogrelate also inhibited the 5-HT-enhanced LR11 mRNA expression in VSMCs. Furthermore, 5-HT suppressed the 7KCHO-induced apoptosis of VSMCs via caspase-3/7-dependent pathway. These findings provide new insights on the changes in the differentiation stage of VSMCs mediated by 5-HT.« less
  • The control of vascular endothelial and muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical cells also synthesize an HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with /sup 125/I-labeled HBGF-I for binding to the HBGF-I cell surfacemore » receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.« less
  • Highlights: Black-Right-Pointing-Pointer TNF-{alpha} augments neointimal hyperplasia in human saphenous vein. Black-Right-Pointing-Pointer TNF-{alpha} induces detrimental effects on endothelial and smooth muscle cell function. Black-Right-Pointing-Pointer Estradiol exerts modulatory effects on TNF-induced vascular cell functions. Black-Right-Pointing-Pointer The modulatory effects of estradiol are discriminatory and cell-type specific. -- Abstract: Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protectivemore » effects of estradiol (E2). We therefore investigated the effects of TNF-{alpha} on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-{alpha} (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-{alpha}, an effect that was abolished by co-culture with E2. TNF-{alpha} increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-{alpha} alone. SV-EC migration was significantly impaired by TNF-{alpha} (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-{alpha} increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-{alpha} potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there was no modulation by E2 in either cell-type. In conclusion, TNF-{alpha} induced SV neointima formation, increased SMC proliferation and migration, impaired SV-EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.« less