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• W2155282657 abstract "Previous studies from our laboratory and others presented evidence that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine can inhibit lipopolysaccharide (LPS)-mediated induction of interleukin-8 (IL-8) in endothelial cells. Using synthetic derivatives of phosphatidylethanolamine, we now demonstrate that phospholipid oxidation products containing α,β-unsaturated carboxylic acids are the most active inhibitors we examined. 5-Keto-6-octendioic acid ester of 2-phosphatidylcholine (KOdiA-PC) was 500-fold more inhibitory than OxPAPC, being active in the nanomolar range. Our studies in human aortic endothelial cells identify one important mechanism of the inhibitory response as involving the activation of neutral sphingomyelinase. There is evidence that Toll-like receptor-4 and other members of the LPS receptor complex must be colocalized to the caveolar/lipid raft region of the cell, where sphingomyelin is enriched, for effective LPS signaling. Previous work from our laboratory suggested that OxPAPC could disrupt this caveolar fraction. These studies present evidence that OxPAPC activates sphingomyelinase, increasing the levels of 16:0, 22:0, and 24:0 ceramide and that the neutral sphingomyelinase inhibitor GW4869 reduces the inhibitory effect of OxPAPC and KOdiA-PC. We also show that cell-permeant C6 ceramide, like OxPAPC, causes the inhibition of LPS-induced IL-8 synthesis and alters caveolin distribution similar to OxPAPC. Together, these data identify a new pathway by which oxidized phospholipids inhibit LPS action involving the activation of neutral sphingomyelinase, resulting in a change in caveolin distribution. Furthermore, we identify specific oxidized phospholipids responsible for this inhibition. Previous studies from our laboratory and others presented evidence that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine can inhibit lipopolysaccharide (LPS)-mediated induction of interleukin-8 (IL-8) in endothelial cells. Using synthetic derivatives of phosphatidylethanolamine, we now demonstrate that phospholipid oxidation products containing α,β-unsaturated carboxylic acids are the most active inhibitors we examined. 5-Keto-6-octendioic acid ester of 2-phosphatidylcholine (KOdiA-PC) was 500-fold more inhibitory than OxPAPC, being active in the nanomolar range. Our studies in human aortic endothelial cells identify one important mechanism of the inhibitory response as involving the activation of neutral sphingomyelinase. There is evidence that Toll-like receptor-4 and other members of the LPS receptor complex must be colocalized to the caveolar/lipid raft region of the cell, where sphingomyelin is enriched, for effective LPS signaling. Previous work from our laboratory suggested that OxPAPC could disrupt this caveolar fraction. These studies present evidence that OxPAPC activates sphingomyelinase, increasing the levels of 16:0, 22:0, and 24:0 ceramide and that the neutral sphingomyelinase inhibitor GW4869 reduces the inhibitory effect of OxPAPC and KOdiA-PC. We also show that cell-permeant C6 ceramide, like OxPAPC, causes the inhibition of LPS-induced IL-8 synthesis and alters caveolin distribution similar to OxPAPC. Together, these data identify a new pathway by which oxidized phospholipids inhibit LPS action involving the activation of neutral sphingomyelinase, resulting in a change in caveolin distribution. Furthermore, we identify specific oxidized phospholipids responsible for this inhibition. Both proinflammatory and anti-inflammatory effects of phospholipid oxidation products on endothelial cells have been demonstrated (1Berliner J.A. Watson A.D. A role for oxidized phospholipids in atherosclerosis..N. Engl. J. Med. 2005; 353: 9-11Crossref PubMed Scopus (217) Google Scholar, 2Bochkov V.N. Leitinger N. Anti-inflammatory properties of lipid oxidation products..J. Mol. Med. 2003; 81: 613-626Crossref PubMed Scopus (74) Google Scholar). The proinflammatory effects lead to chronic monocyte-specific endothelial interactions. Several laboratories have demonstrated that treatment of endothelial cells and macrophages with oxidized LDL and minimally modified low density lipoprotein (MM-LDL) inhibits the acute inflammatory response mediated by lipopolysaccharide (LPS) action (2Bochkov V.N. Leitinger N. Anti-inflammatory properties of lipid oxidation products..J. Mol. Med. 2003; 81: 613-626Crossref PubMed Scopus (74) Google Scholar, 3Leitinger N. Tyner T.R. Oslund L. Rizza C. Subbanagounder G. Lee H. Shih P.T. Mackman N. Tigyi G. Territo M.C. et al.Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils..Proc. Natl. Acad. Sci. USA. 1999; 96: 12010-12015Crossref PubMed Scopus (227) Google Scholar, 4Walton K.A. Cole A.L. Yeh M. Subbanagounder G. Krutzik S.R. Modlin R.L. Lucas R.M. Nakai J. Smart E.J. Vora D.K. et al.Specific phospholipid oxidation products inhibit ligand activation of Toll-like receptors 4 and 2..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1197-1203Crossref PubMed Scopus (166) Google Scholar). Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC), the major active component of MM-LDL, was previously found to inhibit the LPS-mediated induction of E-selectin (3Leitinger N. Tyner T.R. Oslund L. Rizza C. Subbanagounder G. Lee H. Shih P.T. Mackman N. Tigyi G. Territo M.C. et al.Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils..Proc. Natl. Acad. Sci. USA. 1999; 96: 12010-12015Crossref PubMed Scopus (227) Google Scholar) and interleukin-8 (IL-8) synthesis (4Walton K.A. Cole A.L. Yeh M. Subbanagounder G. Krutzik S.R. Modlin R.L. Lucas R.M. Nakai J. Smart E.J. Vora D.K. et al.Specific phospholipid oxidation products inhibit ligand activation of Toll-like receptors 4 and 2..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1197-1203Crossref PubMed Scopus (166) Google Scholar) in human aortic endothelial cells (HAECs) and in human umbilical vein endothelial cells (5Bochkov V.N. Kadl A. Huber J. Gruber F. Binder B.R. Leitinger N. Protective role of phospholipid oxidation products in endotoxin-induced tissue damage..Nature. 2002; 419: 77-81Crossref PubMed Scopus (322) Google Scholar). The effects of OxPAPC were mimicked by oxidized palmitoyl-2-arachidonoyl-sn-glycero-3 phosphatidylethanolamine (OxPAPE), indicating the importance of the oxidation at the sn-2 position of the phospholipid and the similarity in effects of choline- and ethanolamine-containing phospholipid (6Subbanagounder G. Leitinger N. Schwenke D.C. Wong J.W. Lee H. Rizza C. Watson A.D. Faull K.F. Fogelman A.M. Berliner J.A. Determinants of bioactivity of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position..Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2248-2254Crossref PubMed Scopus (197) Google Scholar). For the current studies, we used a more systematic approach to examine the effects of different OxPAPE lipids on LPS action to expand and compare these effects with the more limited studies we had done with OxPAPC. An important goal of the these studies was to identify the most active oxidized phospholipids in inhibiting LPS induction of IL-8.A second goal of the current studies was to examine the mechanism by which OxPAPC inhibits LPS action in HAECs. Several mechanisms have been suggested by which OxPAPC or MM-LDL inhibits the LPS-induced transcription of downstream targets. At the extracellular level, OxPAPC has been shown to block the interaction of LPS with lipopolysaccharide binding protein (LBP) and CD14 when these proteins are bound to dishes (5Bochkov V.N. Kadl A. Huber J. Gruber F. Binder B.R. Leitinger N. Protective role of phospholipid oxidation products in endotoxin-induced tissue damage..Nature. 2002; 419: 77-81Crossref PubMed Scopus (322) Google Scholar). This competition for binding to LBP or CD14 presented as a soluble molecule, as they would be in vivo, is a possible inhibitory mechanism that would act predominantly at the extracellular level. A goal of the current studies was to test the importance of these competitive effects in the OxPAPC inhibition of LPS-induced transcription of IL-8.Studies from our laboratory have demonstrated that oxidized phospholipids also have effects on LPS action at the cellular level, because OxPAPC inhibition of LPS action was observed in cells in which the OxPAPC was removed before LPS addition (4Walton K.A. Cole A.L. Yeh M. Subbanagounder G. Krutzik S.R. Modlin R.L. Lucas R.M. Nakai J. Smart E.J. Vora D.K. et al.Specific phospholipid oxidation products inhibit ligand activation of Toll-like receptors 4 and 2..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1197-1203Crossref PubMed Scopus (166) Google Scholar). These studies suggested that inhibition of the assembly of the LPS receptor complex in a lipid raft/caveolar fraction might be involved in the cellular inhibitory action of OxPAPC. It has been demonstrated previously that activation by LPS requires the recruitment of Toll-like receptor 4 and MD-2 to lipid rafts/caveolar areas of the cell enriched in sphingomyelin (4Walton K.A. Cole A.L. Yeh M. Subbanagounder G. Krutzik S.R. Modlin R.L. Lucas R.M. Nakai J. Smart E.J. Vora D.K. et al.Specific phospholipid oxidation products inhibit ligand activation of Toll-like receptors 4 and 2..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1197-1203Crossref PubMed Scopus (166) Google Scholar, 7Wang P.Y. Kitchens R.L. Munford R.S. Bacterial lipopolysaccharide binds to CD14 in low-density domains of the monocyte-macrophage plasma membrane..J. Inflamm. 1995; 47: 126-137PubMed Google Scholar, 8Triantafilou M. Miyake K. Golenbock D.T. Triantafilou K. Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation..J. Cell Sci. 2002; 115: 2603-2611PubMed Google Scholar, 9Triantafilou M. Morath S. Mackie A. Hartung T. Triantafilou K. Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane..J. Cell Sci. 2004; 117: 4007-4014Crossref PubMed Scopus (131) Google Scholar, 10Dunzendorfer S. Lee H.K. Soldau K. Tobias P.S. Toll-like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS responses..FASEB J. 2004; 18: 1117-1119Crossref PubMed Scopus (122) Google Scholar). Oxidized LDL, MM-LDL, and an OxPAPC component, 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphatidylcholine (POVPC), were shown to trigger an early activation of the sphingomyelin-ceramide pathway, as shown by both sphingomyelin hydrolysis and ceramide formation (11Escargueil-Blanc I. Andrieu-Abadie N. Caspar-Bauguil S. Brossmer R. Levade T. Negre-Salvayre A. Salvayre R. Apoptosis and activation of the sphingomyelin-ceramide pathway induced by oxidized low density lipoproteins are not causally related in ECV-304 endothelial cells..J. Biol. Chem. 1998; 273: 27389-27395Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Loidl A. Sevcsik E. Riesenhuber G. Deigner H.P. Hermetter A. Oxidized phospholipids in minimally modified low density lipoprotein induce apoptotic signaling via activation of acid sphingomyelinase in arterial smooth muscle cells..J. Biol. Chem. 2003; 278: 32921-32928Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 13Chatterjee S. Berliner J.A. Subbanagounder G.G. Bhunia A.K. Koh S. Identification of a biologically active component in minimally oxidized low density lipoprotein (MM-LDL) responsible for aortic smooth muscle cell proliferation..Glycoconj. J. 2004; 20: 331-338Crossref PubMed Scopus (30) Google Scholar). The current studies tested the hypothesis that an increase in sphingomyelinase activity plays a role in the ability of phospholipid oxidation products to inhibit the LPS induction of IL-8 and alter the caveolar/lipid raft fraction.EXPERIMENTAL PROCEDURESReagentsTissue culture media and reagents were obtained from Irvine Scientific, Inc. Fetal bovine serum was obtained from Hyclone. PAPC was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL), or Sigma and oxidized as described previously (14Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. et al.Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo..J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar). Oxidized ethanolamine phospholipids were synthesized as described previously (15Gugiu B.G. Salomon R.G. Total syntheses of bioactive oxidized ethanolamine phospholipids..Org. Lett. 2003; 5: 2797-2799Crossref PubMed Scopus (13) Google Scholar). Mass analysis, liquid chromatography-mass spectrometry, tandem mass spectrometric analysis, and quantitation of oxidized phospholipids were performed as described previously (16Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. Epoxyisoprostane and epoxycyclopentenone phospholipids regulate monocyte chemotactic protein-1 and interleukin-8 synthesis. Formation of these oxidized phospholipids in response to interleukin-1beta..J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). LPS from Escherichia coli O111:B4 (a natural, smooth strain) was obtained from List Biological Laboratories, Inc. Tumor necrosis factor-α (TNF-α) was obtained from R&D Systems. Soluble CD14 (sCD14) was obtained from Dr. Moshe Arditi (Cedar-Sinai, Los Angeles, CA). Recombinant human LBP was obtained from XOMA. C6 ceramide, C6 dihydroceramide, GW4869, and desipramine were obtained from Sigma.Cell cultureHAECs (17Kim J.A. Territo M.C. Wayner E. Carlos T.M. Parhami F. Smith C.W. Haberland M.E. Fogelman A.M. Berliner J.A. Partial characterization of leukocyte binding molecules on endothelial cells induced by minimally oxidized LDL..Arterioscler. Thromb. 1994; 14: 427-433Crossref PubMed Google Scholar) were isolated and cultured as described previously.Cell fractionationCells were fractionated on sucrose gradients and fractions run using Western blotting as described previously (18Parton R.G. Hancock J.F. Caveolin and Ras function..Methods Enzymol. 2001; 333: 172-183Crossref PubMed Scopus (23) Google Scholar). Characterization of these fractions is shown in our previous publication (19Yeh M. Cole A.L. Choi J. Liu Y. Tulchinsky D. Qiao J.H. Fishbein M.C. Dooley A.N. Hovnanian T. Mouilleseaux K. et al.Role for sterol regulatory element-binding protein in activation of endothelial cells by phospholipid oxidation products..Circ. Res. 2004; 95: 780-788Crossref PubMed Scopus (84) Google Scholar). Briefly, 5–100 mm dishes of HAECs were scraped in ice-cold PBS, pelleted, resuspended in 0.5 ml of 0.5 M Na2CO3, and passed through a 21 gauge needle 12 times. Cells were then sonicated on ice three times for 20 s each and then mixed with 0.5 ml of 90% (w/v) sucrose in 25 mM MES, pH 6.5, and 150 mM NaCl. Equal amounts of protein from this lysate-sucrose mixture were transferred to the bottom of a 5 ml thin-walled centrifuge tube (Beckman; No. 344057) and layered sequentially with 1 ml of 35% sucrose-0.25 M Na2CO3, 1 ml of 25% sucrose-0.25 M Na2CO3, and 1 ml of 5% sucrose-0.25 M Na2CO3. Samples were centrifuged for 16 h at 48,000 rpm in an SW-55 rotor at 4°C. Fractions were collected by manually pipetting 12–400 μl fractions from the top of the centrifuge tube and stored at −20°C or assayed immediately. Fractions were run for Western blotting, and the amount of caveolin 1 in fractions 1–3 (which had the highest amount of caveolin 1) was quantitated by densitometry. As an additional check on equal loading, total caveolin levels were also determined in the whole cell lysate.IL-8 assaysAfter treatment, supernatants were collected and assayed for IL-8 or monocyte chemoattractant protein-1/JE using Quantikine ELISA kits (R&D Systems).Sphingomyelinase assaysThe Amplex Red® Sphingomyelinase Assay Kit (Molecular Probes) was used to monitor sphingomyelinase activity. Acid sphingomyelinase activity was measured at acid pH and neutral sphingomyelinase at neutral pH during the sphingomyelin hydrolysis step. Samples were read in a fluorescence microplate reader with absorption and fluorescence emission set at 560 and 590 nm, respectively.Ceramide measurement by electrospray ionization tandem mass spectrometryCells from three 100 mm dishes of HAECs either untreated or treated with OxPAPC were pooled and briefly sonicated. Cells (2.5 mg of protein) and 300 pmol of C8 ceramide were added as a standard for loss during extraction. Lipids were then extracted by a modified Bligh and Dyer method and dried under a stream of argon gas. Using triplicate injections, electrospray ionization tandem mass spectrometry was used to measure different ceramide species from this crude cellular extract as described previously (20Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS)..J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar). A Perkin-Elmer Sciex API III triple quadrupole mass spectrometer fitted with an Ion SprayTM source was used in the positive ion mode for analysis. Spectra were collected by scanning with 0.3 Da step size and a scan speed of ∼6 s at an orifice voltage of 65 V. Tandem mass spectrometry spectra were recorded in the fragment ion mode with argon as the collision gas (CGT = 100) at an orifice voltage of 85 V. The amount of ceramide was calculated as the ratio of the area of the tandem mass spectrometry peak to the area of the internal standard. This experiment was performed on three different cell isolates. For verification of the tandem mass spectrometry identification of particular ceramides, authentic standards were used.StatisticsThe data are presented as means ± SD.RESULTSIdentification of major inhibitory oxidized phospholipidsTo identify the most potent phospholipid oxidation products responsible for inhibiting the LPS induction of IL-8, we examined a series of synthetic phosphatidylethanolamines with palmitic acid residue at the sn-1 position and oxidation epitopes at the sn-2 position (Fig. 1A ). 1-Palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphatidylethanolamine (ON-PE), 9-hydroxy-10-dodecendioic acid ester of lysophosphatidylethanolamine (HDdiA-PE), 1-palmitoyl-2-azelayl-sn-glycero-3-phosphatidylethanolamine (A-PE), and 9-keto-10-dodecendioic acid ester of lysophosphatidylethanolamine (KDdiA-PE) were derived from 1-palmitoyl-2-linoleyl-sn-glycero-3 phosphatidylethanolamine, whereas the other synthetic lipids were derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine. All were tested at a concentration of 4 μg/ml. For comparison, 50 μg/ml OxPAPC and OxPAPE were also tested. Cells were treated with 4 ng/ml LPS in the presence or absence of oxidized phospholipids. After 4 h, IL-8 levels in the medium were determined. 4-Keto-5-heptendioic acid ester of lysophosphatidylethanolamine, 5-keto-6-octendioic acid ester of 2-lysophosphatidylethanolamine (KOdiA-PE), KDdiA-PE, and HDdiA-PE, which contain an α,β-unsaturated carboxylic acid at the sn-2 position, were the most active inhibitors tested, reducing LPS-induced IL-8 synthesis by 80–90% at 4 μg/ml (Fig. 1B). The aldehyde-containing phospholipids 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphatidylethanolamine and ON-PE inhibited LPS action by 50–60%, whereas the carboxylic acid-containing derivatives lacking the α,β-unsaturation, A-PE and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphatidyl ethanolamine, only inhibited LPS action by ∼40%. Because we obtained a high level of inhibition with KOdiA-PE and similar results were seen with phosphatidylcholine and phosphatidylethanolamine derivatives, a dose-response determination was made comparing OxPAPC with KOdiA-PC (Fig. 1C). Although OxPAPC showed significant inhibition at >25 μg/ml, KOdiA-PC showed significant inhibition at 50 ng/ml and thus was 500-fold more active than OxPAPC.Fig. 1.Comparison of the inhibition of lipopolysaccharide (LPS) action by individual oxidized phosphatidylethanolamine species. A: Structures of synthetic oxidized ethanolamine phospholipids used in inhibition studies. PE represents a lysophosphatidylethanolmine (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolmine) residue. B: Inhibition of LPS-induced interleukin-8 (IL-8) synthesis by oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC), oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine (OxPAPE), and seven synthetic oxidized phospholipids shown in A. Human aortic endothelial cells (HAECs) were treated with medium alone (control), 4 ng/ml LPS (L), LPS + 50 μg/ml OxPAPC or OxPAPE, or LPS + 4 μg/ml 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphatidylethanolamine (G-PE), 1-palmitoyl-2-azelayl-sn-glycero-3-phosphatidylethanolamine (A-PE), 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphatidylethanolamine (ON-PE), 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphatidylethanolamine (OV-PE), 4-keto-5-heptendioic acid ester of lysophosphatidylethanolamine (KHdiA-PE), 5-keto-6-octendioic acid ester of 2-lysophosphatidylethanolamine (KOdiA-PE), 9-keto-10-dodecendioic acid ester of lysophosphatidylethanolamine (KDdiA-PE), or 9-hydroxy-10-dodecendioic acid ester of lysophosphatidylethanolamine (HDdiA-PE) for 4 h, and IL-8 levels in the medium were determined. All phospholipids at 4 μg/ml significantly inhibited the ability of LPS to stimulate the synthesis of IL-8 (P < 0.01) compared with cells treated with LPS alone. C: Dose-response curve for the inhibitory effect of OxPAPC and KOdiA-PC on LPS-induced IL-8 synthesis. Cells were treated with control medium or 2 ng/ml LPS with or without the addition of OxPAPC (Ox) or KOdiA-PC (Ko) at the indicated concentrations for 4 h. IL-8 levels in the medium were then determined. Data are given as means ± SD. Each data point was determined in quadruplicate. ** P < 0.01, * P < 0.05 compared with LPS alone. The results shown represent one of three experiments that gave similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 1.Comparison of the inhibition of lipopolysaccharide (LPS) action by individual oxidized phosphatidylethanolamine species. A: Structures of synthetic oxidized ethanolamine phospholipids used in inhibition studies. PE represents a lysophosphatidylethanolmine (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolmine) residue. B: Inhibition of LPS-induced interleukin-8 (IL-8) synthesis by oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC), oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine (OxPAPE), and seven synthetic oxidized phospholipids shown in A. Human aortic endothelial cells (HAECs) were treated with medium alone (control), 4 ng/ml LPS (L), LPS + 50 μg/ml OxPAPC or OxPAPE, or LPS + 4 μg/ml 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphatidylethanolamine (G-PE), 1-palmitoyl-2-azelayl-sn-glycero-3-phosphatidylethanolamine (A-PE), 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphatidylethanolamine (ON-PE), 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphatidylethanolamine (OV-PE), 4-keto-5-heptendioic acid ester of lysophosphatidylethanolamine (KHdiA-PE), 5-keto-6-octendioic acid ester of 2-lysophosphatidylethanolamine (KOdiA-PE), 9-keto-10-dodecendioic acid ester of lysophosphatidylethanolamine (KDdiA-PE), or 9-hydroxy-10-dodecendioic acid ester of lysophosphatidylethanolamine (HDdiA-PE) for 4 h, and IL-8 levels in the medium were determined. All phospholipids at 4 μg/ml significantly inhibited the ability of LPS to stimulate the synthesis of IL-8 (P < 0.01) compared with cells treated with LPS alone. C: Dose-response curve for the inhibitory effect of OxPAPC and KOdiA-PC on LPS-induced IL-8 synthesis. Cells were treated with control medium or 2 ng/ml LPS with or without the addition of OxPAPC (Ox) or KOdiA-PC (Ko) at the indicated concentrations for 4 h. IL-8 levels in the medium were then determined. Data are given as means ± SD. Each data point was determined in quadruplicate. ** P < 0.01, * P < 0.05 compared with LPS alone. The results shown represent one of three experiments that gave similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The role of CD14 and LPB in the inhibitory action of OxPAPCTo determine the role of CD14 and LBP in the inhibition of LPS induction of IL-8 by OxPAPC, we co-treated HAECs with OxPAPC and LPS in the presence and absence of sCD14. Although sCD14 strongly stimulated LPS action, it did not reverse the inhibitory effect of OxPAPC (Fig. 2A ). Concentrations as high as 200 μg/ml sCD14 were unable to prevent the inhibitory effect of 100 ng/ml KOdiA-PE (LPS + sCD14 = 63 ± 4; LPS + sCD14 + KOdiA-PE = 8 ± 0.6 IL-8 pg/ml). Exposure of cells to soluble LBP also significantly increased LPS action but did not inhibit the effect of OxPAPC on LPS action (Fig. 2B).Fig. 2.Effect of soluble CD14 (sCD14; A) and lipopolysaccharide binding protein (LBP; B) on the inhibition of LPS-induced IL-8 synthesis by OxPAPC. HAECs were treated with medium alone (control), 15 μg/ml OxPAPC (Ox), 4 ng/ml LPS, or 15 μg/ml OxPAPC plus 4 ng/ml LPS (Ox + LPS) in the presence of 0, 25, or 50 ng/ml sCD14 (A) or 0, 50, or 100 ng/ml LBP (B) for 4 h. Medium was then used for the IL-8 assay. There was no significant effect of LBP or sCD14 on OxPAPC inhibition. Values shown are means ± SD of quadruplicate determinations. These results are representative of three experiments that gave similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2.Effect of soluble CD14 (sCD14; A) and lipopolysaccharide binding protein (LBP; B) on the inhibition of LPS-induced IL-8 synthesis by OxPAPC. HAECs were treated with medium alone (control), 15 μg/ml OxPAPC (Ox), 4 ng/ml LPS, or 15 μg/ml OxPAPC plus 4 ng/ml LPS (Ox + LPS) in the presence of 0, 25, or 50 ng/ml sCD14 (A) or 0, 50, or 100 ng/ml LBP (B) for 4 h. Medium was then used for the IL-8 assay. There was no significant effect of LBP or sCD14 on OxPAPC inhibition. Values shown are means ± SD of quadruplicate determinations. These results are representative of three experiments that gave similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)OxPAPC-induced sphingomyelinase activation regulates OxPAPC inhibition of LPS induction of IL-8We first determined the ability of OxPAPC to activate sphingomyelinase using an Amplex Red® sphingomyelinase assay; both neutral and acid sphingomyelinase activities were determined. The assays were performed on lysates from HAECs treated for 4 h with control medium, medium containing 50 μg/ml OxPAPC, or medium containing 20 ng/ml TNF-α as a positive control. As shown in Fig. 3A, OxPAPC treatment, as well as TNF-α, significantly increased both neutral and acidic sphingomyelinase activity. We then directly measured levels of ceramide formed in response to OxPAPC. We quantitated the ceramide species produced by OxPAPC treatment using electrospray ionization tandem mass spectrometry. OxPAPC treatment (50 μg/ml for 4 h) significantly increased C16:0, C22:0, and C24:0 (Fig. 3B), whereas C24:1 was unchanged (data not shown). To determine whether these changes in enzymatic activity play a role in the OxPAPC inhibition of LPS action, we used specific inhibitors of neutral sphingomyelinase (GW4869; 15 μM) and acid sphingomyelinase (desipramine; 10 μM). Assays were performed to verify that the concentrations of inhibitor used were sufficient to inhibit the induction of sphingomyelinase activity by OxPAPC. For these experiments, cells were pretreated for 1 h with GW4869, desipramine, or vehicle control before treatment with control medium or medium containing 50 μg/ml OxPAPC for 4 h. The OxPAPC-induced neutral sphingomyelinase activity was reduced by >90% with GW4869 but not with desipramine, whereas the acid sphingomyelinase activity was reduced by 90% with desipramine but not with GW4869 (data not shown). We next tested the effect of these inhibitors on the ability of OxPAPC to inhibit LPS induction of IL-8. Cells were pretreated for 1 h with vehicle control, GW4869, or desipramine. Then, control medium, OxPAPC, OxPAPC + LPS, KOdiA-PC, or KOdiA-PC + LPS was added to the wells. After 4 h, medium was collected and IL-8 levels were determined (Fig. 4A ). GW4869 significantly inhibited the ability of OxPAPC and KOdiA-PC to decrease the LPS induction of IL-8, but desipramine was ineffective (Fig. 4B).Fig. 3.Effect of OxPAPC on neutral and acidic sphingomyelinase activity (A) and ceramide production (B). A: Cell lysates from HAECs treated with medium alone (control), 50 μg/ml O" @default.
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