Acyl chain-dependent effect of lysophosphatidylcholine on endothelial prostacyclin production
2010; Elsevier BV; Volume: 51; Issue: 10 Linguagem: Inglês
10.1194/jlr.m006536
ISSN1539-7262
AutoresMonika Riederer, Pauli J. Ojala, Andelko Hrzenjak, Wolfgang F. Graier, Roland Malli, Michaela Tritscher, Martin Hermansson, Bernhard Watzer, Horst Schweer, Gernot Desoyé, Ákos Heinemann, Saša Frank,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoPreviously we identified palmitoyl-lysophosphatidylcholine (16:0 LPC), linoleoyl-LPC (18:2 LPC), arachidonoyl-LPC (20:4 LPC), and oleoyl-LPC (18:1 LPC) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein. In the present study, the impact of those LPC on prostacyclin (PGI2) production was examined in vitro in primary human aortic endothelial cells (HAEC) and in vivo in mice. Although 18:2 LPC was inactive, 16:0, 18:1, and 20:4 LPC induced PGI2 production in HAEC by 1.4-, 3-, and 8.3-fold, respectively. LPC-elicited 6-keto PGF1α formation depended on both cyclooxygenase (COX)-1 and COX-2 and on the activity of cytosolic phospholipase type IVA (cPLA2). The LPC-induced, cPLA2-dependent 14C-arachidonic acid (AA) release was increased 4.5-fold with 16:0, 2-fold with 18:1, and 2.7-fold with 20:4 LPC, respectively, and related to the ability of LPC to increase cytosolic Ca2+ concentration. In vivo, LPC increased 6-keto PGF1α concentration in mouse plasma with a similar order of potency as found in HAEC. Our results indicate that the tested LPC species are capable of eliciting production of PGI2, whereby the efficacy and the relative contribution of underlying mechanisms are strongly related to acyl-chain length and degree of saturation. Previously we identified palmitoyl-lysophosphatidylcholine (16:0 LPC), linoleoyl-LPC (18:2 LPC), arachidonoyl-LPC (20:4 LPC), and oleoyl-LPC (18:1 LPC) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein. In the present study, the impact of those LPC on prostacyclin (PGI2) production was examined in vitro in primary human aortic endothelial cells (HAEC) and in vivo in mice. Although 18:2 LPC was inactive, 16:0, 18:1, and 20:4 LPC induced PGI2 production in HAEC by 1.4-, 3-, and 8.3-fold, respectively. LPC-elicited 6-keto PGF1α formation depended on both cyclooxygenase (COX)-1 and COX-2 and on the activity of cytosolic phospholipase type IVA (cPLA2). The LPC-induced, cPLA2-dependent 14C-arachidonic acid (AA) release was increased 4.5-fold with 16:0, 2-fold with 18:1, and 2.7-fold with 20:4 LPC, respectively, and related to the ability of LPC to increase cytosolic Ca2+ concentration. In vivo, LPC increased 6-keto PGF1α concentration in mouse plasma with a similar order of potency as found in HAEC. Our results indicate that the tested LPC species are capable of eliciting production of PGI2, whereby the efficacy and the relative contribution of underlying mechanisms are strongly related to acyl-chain length and degree of saturation. Lysophosphatidylcholine (LPC) is a bioactive phospholipid generated primarily by the action of phospholipase A2 (PLA2) enzymes on plasma membrane- and lipoprotein-phosphatidylcholine (PC) containing saturated fatty acid (FA) at the sn-1 and mostly unsaturated FA at the sn-2 position (1Ruiperez V. Casas J. Balboa M.A. Balsinde J. Group V phospholipase A2-derived lysophosphatidylcholine mediates cyclooxygenase-2 induction in lipopolysaccharide-stimulated macrophages.J. Immunol. 2007; 179: 631-638Crossref PubMed Scopus (42) Google Scholar, 2Sato H. Kato R. Isogai Y. Saka G. Ohtsuki M. Taketomi Y. Yamamoto K. Tsutsumi K. Yamada J. Masuda S. et al.Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis.J. Biol. Chem. 2008; 283: 33483-33497Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 3Zalewski A. Macphee C. Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology, and possible therapeutic target.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 923-931Crossref PubMed Scopus (405) Google Scholar). LPC can also be produced as a by-product by lecithin cholesterol acyltransferase (LCAT) in high-density lipoprotein (HDL) (4Rousset X. Vaisman B. Amar M. Sethi A.A. Remaley A.T. Lecithin:cholesterol acyltransferase—from biochemistry to role in cardiovascular disease.Curr. Opin. Endocrinol. Diabetes Obes. 2009; 16: 163-171Crossref PubMed Scopus (132) Google Scholar) as well as from oxidation of low-density lipoprotein (LDL) (5Parthasarathy S. Steinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein.Proc. Natl. Acad. Sci. USA. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar) and by endothelial lipase (EL).The physiological concentrations of LPC in body fluids is high, around 150 µM (6Rabini R.A. Galassi R. Fumelli P. Dousset N. Solera M.L. Valdiguie P. Curatola G. Ferretti G. Taus M. Mazzanti L. Reduced Na(+)-K(+)-ATPase activity and plasma lysophosphatidylcholine concentrations in diabetic patients.Diabetes. 1994; 43: 915-919Crossref PubMed Scopus (79) Google Scholar, 7Subbaiah P.V. Chen C.H. Bagdade J.D. Albers J.J. Substrate specificity of plasma lysolecithin acyltransferase and the molecular species of lecithin formed by the reaction.J. Biol. Chem. 1985; 260: 5308-5314Abstract Full Text PDF PubMed Google Scholar), with even millimolar levels in hyperlipidemic subjects (8Chen L. Liang B. Froese D.E. Liu S. Wong J.T. Tran K. Hatch G.M. Mymin D. Kroeger E.A. Man R.Y. et al.Oxidative modification of low density lipoprotein in normal and hyperlipidemic patients: effect of lysophosphatidylcholine composition on vascular relaxation.J. Lipid Res. 1997; 38: 546-553Abstract Full Text PDF PubMed Google Scholar). LPC in plasma is distributed between albumin or some other carrier serum proteins (9Ojala P.J. Hermansson M. Tolvanen M. Polvinen K. Hirvonen T. Impola U. Jauhiainen M. Somerharju P. Parkkinen J. Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine.Biochemistry. 2006; 45: 14021-14031Crossref PubMed Scopus (50) Google Scholar, 10Croset M. Brossard N. Polette A. Lagarde M. Characterization of plasma unsaturated lysophosphatidylcholines in human and rat.Biochem. J. 2000; 345: 61-67Crossref PubMed Scopus (0) Google Scholar) and lipoproteins (11Marathe G.K. Silva A.R. de Castro Faria Neto H.C. Tjoelker L.W. Prescott S.M. Zimmerman G.A. McIntyre T.M. Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids.J. Lipid Res. 2001; 42: 1430-1437Abstract Full Text Full Text PDF PubMed Google Scholar), with the likely existence of minute amounts of free LPC. This free LPC may transiently arise during an excessive lipolysis when the concentrations of FA and LPC locally exceed the binding capacity of albumin. Under such conditions, LPC partitions transiently to lipoproteins, from where it is rapidly delivered to cells. After uptake into cells, free LPC may be reacylated to yield PC (12Stoll L.L. Oskarsson H.J. Spector A.A. Interaction of lysophosphatidylcholine with aortic endothelial cells.Am. J. Physiol. 1992; 262: H1853-H1860PubMed Google Scholar) or deacylated to provide FA and choline (10Croset M. Brossard N. Polette A. Lagarde M. Characterization of plasma unsaturated lysophosphatidylcholines in human and rat.Biochem. J. 2000; 345: 61-67Crossref PubMed Scopus (0) Google Scholar). Despite abundant evidence for the capacity of the free LPC to increase cytosolic Ca2+ (13Meyer zu Heringdorf D. Jakobs K.H. Lysophospholipid receptors: signalling, pharmacology and regulation by lysophospholipid metabolism.Biochim. Biophys. Acta. 2007; 1768: 923-940Crossref PubMed Scopus (305) Google Scholar, 14Wong J.T. Tran K. Pierce G.N. Chan A.C. K.O. Choy P.C. Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells.J. Biol. Chem. 1998; 273: 6830-6836Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 15Inoue N. Hirata K. Yamada M. Hamamori Y. Matsuda Y. Akita H. Yokoyama M. Lysophosphatidylcholine inhibits bradykinin-induced phosphoinositide hydrolysis and calcium transients in cultured bovine aortic endothelial cells.Circ. Res. 1992; 71: 1410-1421Crossref PubMed Scopus (96) Google Scholar, 16Yokoyama K. Ishibashi T. Ohkawara H. Kimura J. Matsuoka I. Sakamoto T. Nagata K. Sugimoto K. Sakurada S. Maruyama Y. HMG-CoA reductase inhibitors suppress intracellular calcium mobilization and membrane current induced by lysophosphatidylcholine in endothelial cells.Circulation. 2002; 105: 962-967Crossref PubMed Scopus (64) Google Scholar) and activate numerous signaling pathways (17Prokazova N.V. Zvezdina N.D. Korotaeva A.A. Effect of lysophosphatidylcholine on transmembrane signal transduction.Biochemistry-Russia. 1998; 63: 31-37PubMed Google Scholar, 18Bassa B.V. Roh D.D. Vaziri N.D. Kirschenbaum M.A. Kamanna V.S. Lysophosphatidylcholine activates mesangial cell PKC and MAP kinase by PLCgamma-1 and tyrosine kinase-Ras pathways.Am. J. Physiol. 1999; 277: F328-F337PubMed Google Scholar) resulting in an enhanced expression of inflammatory molecules (19Kume N. Cybulsky M.I. Gimbrone Jr, M.A. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells.J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (720) Google Scholar, 20Aiyar N. Disa J. Ao Z. Ju H. Nerurkar S. Willette R.N. Macphee C.H. Johns D.G. Douglas S.A. Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells.Mol. Cell. Biochem. 2007; 295: 113-120Crossref PubMed Scopus (104) Google Scholar) and prostanoids (EC) (14Wong J.T. Tran K. Pierce G.N. Chan A.C. K.O. Choy P.C. Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells.J. Biol. Chem. 1998; 273: 6830-6836Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21Zembowicz A. Jones S.L. Wu K.K. Induction of cyclooxygenase-2 in human umbilical vein endothelial cells by lysophosphatidylcholine.J. Clin. Invest. 1995; 96: 1688-1692Crossref PubMed Scopus (88) Google Scholar, 22Rikitake Y. Hirata K. Kawashima S. Takeuchi S. Shimokawa Y. Kojima Y. Inoue N. Yokoyama M. Signaling mechanism underlying COX-2 induction by lysophosphatidylcholine.Biochem. Biophys. Res. Commun. 2001; 281: 1291-1297Crossref PubMed Scopus (56) Google Scholar), the role of the putative LPC receptors is still a matter of debate.It is largely accepted that LPC may act via a subset of G protein-coupled receptors (GPCR), including GPR4, G2A (G2 accumulation), OGR1 (ovarian cancer G protein-coupled receptor 1), and TDAG8 (T cell death-associated gene 8), that are sensitive to both LPC and protons (23Ludwig M.G. Vanek M. Guerini D. Gasser J.A. Jones C.E. Junker U. Hofstetter H. Wolf R.M. Seuwen K. Proton-sensing G-protein-coupled receptors.Nature. 2003; 425: 93-98Crossref PubMed Scopus (513) Google Scholar, 24Tomura H. Mogi C. Sato K. Okajima F. Proton-sensing and lysolipid-sensitive G-protein-coupled receptors: a novel type of multi-functional receptors.Cell. Signal. 2005; 17: 1466-1476Crossref PubMed Scopus (145) Google Scholar). Of these receptors, GPR4 shows the widest tissue distribution and is abundantly expressed in vascular endothelial cells where its expression is enhanced by inflammation (25Lum H. Qiao J. Walter R.J. Huang F. Subbaiah P.V. Kim K.S. Holian O. Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells.Am. J. Physiol. Heart Circ. Physiol. 2003; 285: H1786-H1789Crossref PubMed Scopus (54) Google Scholar). In contrast, G2A, whose cell surface expression and stabilization are enhanced by LPC (26Wang L. Radu C.G. Yang L.V. Bentolila L.A. Riedinger M. Witte O.N. Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G protein-coupled receptor G2A.Mol. Biol. Cell. 2005; 16: 2234-2247Crossref PubMed Scopus (69) Google Scholar) but whose proton-induced actions are antagonized by LPC (27Murakami N. Yokomizo T. Okuno T. Shimizu T. G2A is a proton-sensing G-protein-coupled receptor antagonized by lysophosphatidylcholine.J. Biol. Chem. 2004; 279: 42484-42491Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), is mainly expressed in lymphoid tissue, lymphocytes, and macrophages (24Tomura H. Mogi C. Sato K. Okajima F. Proton-sensing and lysolipid-sensitive G-protein-coupled receptors: a novel type of multi-functional receptors.Cell. Signal. 2005; 17: 1466-1476Crossref PubMed Scopus (145) Google Scholar). Although the initial papers were retracted due to failure to reproduce the binding of LPC to GPR4 and G2A, respectively, several studies have shown that LPC stimulates a variety of cellular activities dependent on GPR4 (25Lum H. Qiao J. Walter R.J. Huang F. Subbaiah P.V. Kim K.S. Holian O. Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells.Am. J. Physiol. Heart Circ. Physiol. 2003; 285: H1786-H1789Crossref PubMed Scopus (54) Google Scholar, 28Zou Y. Kim C.H. Chung J.H. Kim J.Y. Chung S.W. Kim M.K. Im D.S. Lee J. Yu B.P. Chung H.Y. Upregulation of endothelial adhesion molecules by lysophosphatidylcholine. Involvement of G protein-coupled receptor GPR4.FEBS J. 2007; 274: 2573-2584Crossref PubMed Scopus (27) Google Scholar, 29Qiao J. Huang F. Naikawadi R.P. Kim K.S. Said T. Lum H. Lysophosphatidylcholine impairs endothelial barrier function through the G protein-coupled receptor GPR4.Am. J. Physiol. Lung Cell. Mol. Physiol. 2006; 291: L91-L101Crossref PubMed Scopus (97) Google Scholar). However, some studies that failed to observe the LPC actions in GPR4-expressing cells provided evidence for the capacity of GPR4 to sense extracellular protons, resulting in G-protein-mediated intracellular signaling and cAMP accumulation (30Sin W.C. Zhang Y. Zhong W. Adhikarakunnathu S. Powers S. Hoey T. An S. Yang J. G protein-coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers.Oncogene. 2004; 23: 6299-6303Crossref PubMed Scopus (80) Google Scholar, 31Bektas M. Barak L.S. Jolly P.S. Liu H. Lynch K.R. Lacana E. Suhr K.B. Milstien S. Spiegel S. The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner.Biochemistry. 2003; 42: 12181-12191Crossref PubMed Scopus (62) Google Scholar, 32Tobo M. Tomura H. Mogi C. Wang J.Q. Liu J.P. Komachi M. Damirin A. Kimura T. Murata N. Kurose H. et al.Previously postulated "ligand-independent" signaling of GPR4 is mediated through proton-sensing mechanisms.Cell. Signal. 2007; 19: 1745-1753Crossref PubMed Scopus (68) Google Scholar).Prostanoid biosynthesis involves oxidation and subsequent isomerization of unesterified arachidonic acid (AA). The initial step of this metabolic pathway is the stimulus-induced release of AA from membrane phospholipids by phospholipase A2 (PLA2) enzymes, principally Ca2+-dependent cytosolic PLA2IVA (cPLA2), followed by conversion to prostaglandin H2 (PGH2) by either cyclooxygenase (COX)-1 or COX-2, both constitutively expressed by vascular endothelial cells (EC) (33Ghosh M. Wang H. Ai Y. Romeo E. Luyendyk J.P. Peters J.M. Mackman N. Dey S.K. Hla T. COX-2 suppresses tissue factor expression via endocannabinoid-directed PPARdelta activation.J. Exp. Med. 2007; 204: 2053-2061Crossref PubMed Scopus (60) Google Scholar, 34McAdam B.F. Catella-Lawson F. Mardini I.A. Kapoor S. Lawson J.A. FitzGerald G.A. Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2.Proc. Natl. Acad. Sci. USA. 1999; 96 ([Erratum. 1999. Proc. Natl. Acad. Sci. USA.96: 5890.]): 272-277Crossref PubMed Scopus (1182) Google Scholar, 35Alfranca A. Iniguez M.A. Fresno M. Redondo J.M. Prostanoid signal transduction and gene expression in the endothelium: role in cardiovascular diseases.Cardiovasc. Res. 2006; 70: 446-456Crossref PubMed Scopus (84) Google Scholar). PGH2 is then converted to various prostanoids by the respective terminal prostanoid synthases (36Dogne J.M. Hanson J. Pratico D. Thromboxane, prostacyclin and isoprostanes: therapeutic targets in atherogenesis.Trends Pharmacol. Sci. 2005; 26: 639-644Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 37Ueno N. Takegoshi Y. Kamei D. Kudo I. Murakami M. Coupling between cyclooxygenases and terminal prostanoid synthases.Biochem. Biophys. Res. Commun. 2005; 338: 70-76Crossref PubMed Scopus (92) Google Scholar).Endothelial lipase is a phospholipase localized on the surface of vascular endothelial cells (38Hirata K. Dichek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T. Cloning of a unique lipase from endothelial cells extends the lipase gene family.J. Biol. Chem. 1999; 274: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 39Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism.Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (415) Google Scholar). We demonstrated previously that EL, by cleaving HDL-PC, generates substantial amounts of LPC 16:0, 18:1, 18:2, and 20:4, respectively (40Gauster M. Rechberger G. Sovic A. Horl G. Steyrer E. Sattler W. Frank S. Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine.J. Lipid Res. 2005; 46: 1517-1525Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). As studies investigating the impact of LPC on prostanoid production have so far used 16:0 LPC exclusively (1Ruiperez V. Casas J. Balboa M.A. Balsinde J. Group V phospholipase A2-derived lysophosphatidylcholine mediates cyclooxygenase-2 induction in lipopolysaccharide-stimulated macrophages.J. Immunol. 2007; 179: 631-638Crossref PubMed Scopus (42) Google Scholar, 14Wong J.T. Tran K. Pierce G.N. Chan A.C. K.O. Choy P.C. Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells.J. Biol. Chem. 1998; 273: 6830-6836Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21Zembowicz A. Jones S.L. Wu K.K. Induction of cyclooxygenase-2 in human umbilical vein endothelial cells by lysophosphatidylcholine.J. Clin. Invest. 1995; 96: 1688-1692Crossref PubMed Scopus (88) Google Scholar, 22Rikitake Y. Hirata K. Kawashima S. Takeuchi S. Shimokawa Y. Kojima Y. Inoue N. Yokoyama M. Signaling mechanism underlying COX-2 induction by lysophosphatidylcholine.Biochem. Biophys. Res. Commun. 2001; 281: 1291-1297Crossref PubMed Scopus (56) Google Scholar), nothing is known about the impact of the length and degree of saturation of the LPC-acyl chain on prostanoid production. Therefore, the aim of the present study was to assess the impact of those LPC, found to be abundantly generated by EL, on PGI2 production in vitro in HAEC and in vivo in mice.MATERIALS AND METHODSLPCAll LPC was purchased from Avanti Polar Lipids, and additional preparations of 18:2 and 20:4 were generated as described elsewhere (41Ojala P.J. Hirvonen T.E. Hermansson M. Somerharju P. Parkkinen J. Acyl chain-dependent effect of lysophosphatidylcholine on human neutrophils.J. Leukoc. Biol. 2007; 82: 1501-1509Crossref PubMed Scopus (73) Google Scholar). LPC was dissolved and stored at −20°C in chloroform/methanol under argon atmosphere. For experiments, required amounts of LPC were dried under a stream of nitrogen or argon and redissolved in PBS (pH 7.4) for cell culture experiments or in pyrogen-free saline for in vivo experiments.Cell cultureHuman primary aortic endothelial cells (HAEC) were obtained from Lonza and maintained in endothelial cell growth medium [EGM-MV Bullet Kit = EBM medium + growth supplements + FCS (Lonza)] supplemented with 50 IU/ml penicillin and 50 µg/ml streptomycin. Cells were cultured in gelatin coated dishes at 37°C in a 5% CO2 humidified atmosphere and were used for experiments from passage 5 to 10. Cells were seeded (75000/well) in 12-well plates 48 h before exposure to LPC.LPC treatment of HAECInitial time- and concentration-dependent experiments revealed that in a serum-free medium in the absence of BSA, all tested LPC at concentrations up to 10 µM were not toxic to HAEC (for incubations up to 8 h), as determined by monitoring the release of lactate dehydrogenase (LDH) using the cytotoxicity detection kit (LDH) (Roche, Mannheim, Germany).At 48 h after plating, cells were washed with PBS and incubated with EBM medium without supplements and serum for 3 h. This medium is referred to as "serum-free medium" throughout the article. Thereafter, medium was removed and replaced with fresh serum-free medium supplemented with different concentrations of LPC alone or in some experiments along with AA or A23187. In some experiments, as indicated in the respective figure legends, LPC was applied in complex with BSA at a molar ratio of 1:1 and 5:1, respectively. Medium was collected in prechilled tubes at different time points following exposure to LPC, spun to remove cells, and used for lactate dehydrogenase (LDH) assay and measurement of prostanoids. Cells were washed with PBS and lysed in respective buffers for isolation of RNA or proteins.Pharmacological inhibitorsDuring final 30 min of incubation with serum-free medium prior to exposure to LPC as well as during exposure to LPC, HAEC were treated with respective pharmacological inhibitors (all from Calbiochem) or vehicle (DMSO). Specific COX-1 inhibitor SC-560 (100 nM), COX-2 inhibitor NS-398 (20 µM), and cPLA2 inhibitor (1 µM) [(N-{(2S,4R)-4-(biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide, HCl)] were applied.Quantitative real-time PCRRNA of cell extracts was isolated using the peqGOLD Total RNA Kit (Peqlab-biotechnology, Erlangen, Germany) according to the manufacturer's protocol, including on column DNase digestion. Then 1.5 µg of RNA were reverse transcribed using the Archive cDNA Kit (Applied Biosystems, Foster City, CA) and 0.7 U of an RNase Inhibitor (Qiagen, Hilden, Germany). RT-PCR analysis was performed in 384-well plates in a total volume of 4 µl containing 2 ng of original total RNA using the QantiFast SYBR green RT-PCR kit (Qiagen) and validated QuantiTect Primer Assays (Qiagen) according to the manufacturer's instructions for Light Cycler 480 instruments (Roche Diagnostics). In brief, after the initial heat activation step at 95°C for 5 min, cycling conditions consisted of 40 cycles of denaturation at 95°C for 10 s and combined annealing and extension at 60°C for 30 s. The PCR efficiency of the target and housekeeping genes was determined by cDNA dilution series prepared from an untreated sample, and results were accordingly efficiency-corrected with the LightCycler Relative Quantification software (Roche Diagnostics, Basel, Switzerland). mRNA levels of COX-1 and COX-2 (Primer Assays QT00210280 and QT00040586) were normalized to human β-2-microglobulin (Primer Assay QT01665006) and expressed as relative ratio (ΔΔ Ct). All samples were assayed in duplicate, and the average value was used for quantification.Prostanoid profiling by GC-MS/MSThe spectrum of prostanoids produced by HAEC under basal conditions was determined by gas chromatography- tandem mass spectrometry (GC-MS/MS) as described previously (42Schweer H. Watzer B. Seyberth H.W. Determination of seven prostanoids in 1 ml of urine by gas chromatography-negative ion chemical ionization triple stage quadrupole mass spectrometry.J. Chromatogr. 1994; 652: 221-227Crossref PubMed Scopus (71) Google Scholar). In brief, 10 µl deuterated internal standard mixture (about 1 ng of each PG) was added to 0.2–1 ml of each sample. After acidification with formic acid (5%, v/v) to pH 2.6, a solution of 0.1 g methoxyamine hydrochloride in 1 ml sodium acetate (1.5 M, pH 5.0) was added. The prostanoid derivates were extracted with ethyl acetate/hexane (7:3, v/v). After evaporation, the residues were esterified with a reaction mixture of acetone (80 µl), diisopropylamine (7 µl), and pentafluorobenzyl bromide (6 µl) for 10 min at 40°C. Dried samples were then applied to TLC and developed in ethyl acetate/hexane (9:1, v/v). The target zones were scraped off (Rf = 0.03–0.39) and extracted with ethyl acetate. The extracts were then evaporated, derivatized twice with 10 µl BSTFA at 60°C, and analyzed using GC-MS/MS.6-keto PGF1α measurements by EIA6-keto PGF1α was measured in cell culture media and 5-fold diluted mouse plasma by a correlate-EIA kit (Cayman, Ann Arbor, MI) according to the manufacturer's protocol. Protein content of cell culture wells was initially determined to be equal for all treatments.14C-Arachidonic acid and 14C-6-keto PGF1α releaseCells were labeled with 14C-AA (4 µM, spec. activity 58 mCi/mmol) in complete medium for 20 h. Unbound 14C-AA was removed by excessive washing in PBS supplemented with 1% BSA. After incubation in serum-free medium for 3 h, and facultative preincubation with 1 µM cPLA2 inhibitor for 30 min, cells were exposed to 50 µM LPC + 3.7 µM BSA (± cPLA2 inhibitor) for 20 min. Cell media were then collected, spun to remove cells, and immediately frozen at −70°C until extraction. Cells were washed with PBS and lysed in 0.3 M NaOH/0.1% SDS. Aliquots of the cell lysates were mixed with scintillation cocktail, and the radioactivity was determined on a β-counter (Beckman). Acidified media were extracted with 2 vol of hexane/isopropanol (3:2, v/v), evaporated in the SpeedVac, and redissolved in chloroform, followed by TLC using ethylacetate/isooctane/water/acetic acid (11:5:10:2, v/v) as a mobile phase. The signals corresponding to 14C-AA and 14C-6-keto PGF1α were visualized upon exposure of the TLC plates to a tritium screen (GE Healthcare) on the STORM imager. Quantification was performed by densitometric volume report analysis or by liquid scintillation counting of cut out TLC spots corresponding to the comigrating AA- and 6-keto PGF1α-standard [visualized by spraying with primulin (0.01%, w/v) in acetone/water (60:40, v/v) and subsequent UV detection]. The amounts of 14C-AA and 14C-6-keto PGF1α released into medium were normalized to total cellular radioactivity measured by scintillation counting of cell lysates.Western blottingFor quantification of COX-2 protein, three 12-well plates of HAEC treated with LPC or PBS were washed with PBS and lysed in 50 µl of loading buffer [20% (w/v) glycerol, 5% (w/v) SDS, 0.15% (w/v) bromophenol blue, 63 mmol/l Tris-HCl (pH 6.8), and 5% (v/v) β-mercaptoethanol] and boiled for 10 min. Then 40 µl of the lysate were subjected to each lane and analyzed by SDS-PAGE (10% gel) and subsequent immunoblotting using goat anti-COX-2 (Santa Cruz Biotechnology, M19, sc-1747, 1:100) and the HRP-labeled rabbit anti-goat IgG (Dako) as a secondary antibody. Protein signals were detected by ECL assay, and the intensity was normalized to actin (Pierce, ms Anti-Actin IgM: 1:4000) using densitometry.Measurements of [Ca2+]iHAEC were seeded in 6-well plates. At 48 h after plating, cells were serum starved for 2 h and then loaded with 2 µM fura-2/AM for 60 min. After washing, cells were trypsinized and resuspended either in calcium buffer (138 mM NaCl, 1 mM MgCl2, 5 mM KCl, 10 mM Hepes, 10 mM Glucose, pH 7.4) or EGTA buffer (0.1 mM EGTA, 138 mM NaCl, 1 mM MgCl2, 5 mM KCl, 10 mM Hepes, 10 mM Glucose, pH 7.4). The ratio of fura-2 fluorescence intensity at the two excitation wavelengths (340/380 ratio) was monitored spectrophotometrically in a stirring cuvette before and after the injection of 10 µM LPC. In some experiments, cells were resuspended in EGTA buffer followed by a preincubation with either 100 µM 2-APB for 2 min or with 2 µM U73122 for 5 min before the injection of LPC.Experiments in miceFollowing a 6 h fasting period, 10–12-weeks-old male C57Bl/6J mice (4–7 per group) were treated with 20 mg/kg LPC in 0.9% NaCl (or NaCl alone) via tail vein injection. After 20 h, blood was collected from the retro-orbital plexus into tubes containing indomethacin and EDTA, centrifuged immediately, and stored at −70°C for measurements of 6-keto PGF1α. For tail-vein injection and bleeding, mice were anesthetized with Isoflurane (Pharmacia and Upjohn SA, Guyancourt, France). Animal experiments were conducted in conformity with the Public Health Service Policy on Human Care and Use of Laboratory Animals and were approved by the Austrian Ministry of Science and Research according to the Regulations for Animal Experimentation.Statistical analysisCell culture experiments were performed at least three times and values are expressed as mean plus SEM. Statistical significance was determined by the Student's unpaired t-test (two-tailed) with application of Welch's correction, where required. Group differences were considered significant for P < 0.05 (∗), P < 0.01 (∗∗), and P < 0.001 (∗∗∗).RESULTSLPC induces PGI2 production in HAECAs determined by GC-MS/MS analysis, HAEC secrete 6-keto PGF1α (a stable degradation product of PGI2), thromboxane B2 (TxB2; a stable degradation product of TxA2), PGF2α, and PGE2 (Fig. 1A).To examine the impact of LPC on endothelial PGI2 production, HAEC were incubated with 10 µM LPC followed by EIA-based quantification of 6-keto PGF1α. As shown in Fig. 1B, 16:0 LPC increased slightly (1.4-fold) and not statistically significantly the formation of 6-keto PGF1α relative to the PBS control. The increases in 6-keto PGF1α elicited by 18:1 LPC and 20:4 LPC were much more pronounced at 3- and 8.3-fold, respectively. 18:2 LPC did not have any impact on 6-keto PGF1α production (supplementary Fig. I) and was not further studied.LPC-induced 6-keto PGF1α production is mediated by COXLPC upregulates COX-2 mRNA but not COX-2 protein.The ability of both specific COX-1 (SC-560; 100 nM) and COX-2 (NS-398; 20 µM) inhibitors to prevent the LPC-mediated inc
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