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• W2005651799 abstract "Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A2 (cPLA2). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor α (PPARα) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARα null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA2 and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases. Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A2 (cPLA2). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor α (PPARα) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARα null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA2 and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases. One of the earliest steps in the development of the atherosclerotic lesion is the adhesion of monocytes to endothelial cells of the vessel wall (1Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4735) Google Scholar). Previously, it was demonstrated that, if stimulated with minimally modified low density lipoprotein (MM-LDL), endothelial cells are activated to specifically bind monocytes but not neutrophils (2Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions.J. Clin. Invest. 1990; 85: 1260-1266Crossref PubMed Scopus (769) Google Scholar). This specificity toward mononuclear cells was subsequently observed with the activation of endothelial cells by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) (3Berliner J. Leitinger N. Watson A. Huber J. Fogelman A. Navab M. Oxidized lipids in atherogenesis: formation, destruction and action.Thromb. Haemost. 1997; 78: 195-199Crossref PubMed Scopus (126) Google Scholar), implicating lipid oxidation products as culprits in chronic inflammation. One biologically active oxidized component phospholipid in OxPAPC is 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), which essentially mimics the actions of MM-LDL and OxPAPC. POVPC has also been found in MM-LDL, atherosclerotic lesions of animals and humans, and membranes of apoptotic cells (4Watson 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 (692) Google Scholar, 5Subbanagounder 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 (199) Google Scholar, 6Huber J. Vales A. Mitulovic G. Blumer M. Schmid R. Witztum J.L. Binder B.R. Leitinger N. Oxidized membrane vesicles and blebs from apoptotic cells contain biologically active oxidized phospholipids that induce monocyte-endothelial interactions.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 101-107Crossref PubMed Scopus (248) Google Scholar, 7Chang M.K. Binder C.J. Miller Y.I. Subbanagounder G. Silverman G.J. Berliner J.A. Witztum J.L. Apoptotic cells with oxidation-specific epitopes are immunogenic and proinflammatory.J. Exp. Med. 2004; 200: 1359-1370Crossref PubMed Scopus (283) Google Scholar). Monocyte binding induced by oxidized phospholipids in human aortic endothelial cells (HAECs) was shown to be independent of nuclear factor-κB (NF-κB) activation or increased expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) (8Huber J. Boechzelt H. Karten B. Surboeck M. Bochkov V.N. Binder B.R. Sattler W. Leitinger N. Oxidized cholesteryl linoleates stimulate endothelial cells to bind monocytes via the extracellular signal-regulated kinase 1/2 pathway.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 581-586Crossref PubMed Scopus (43) Google Scholar, 9Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Oxidized phospholipids stimulate tissue factor expression in human endothelial cells via activation of ERK/EGR-1 and Ca(++)/NFAT.Blood. 2002; 99: 199-206Crossref PubMed Scopus (180) Google Scholar, 10Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar), but it involves the activation of an α5β1 integrin and increased deposition of the connecting segment-1 (CS-1) domain of fibronectin on the luminal surface of endothelial cells (11Shih P.T. Elices M.J. Fang Z.T. Ugarova T.P. Strahl D. Territo M.C. Frank J.S. Kovach N.L. Cabanas C. Berliner J.A. et al.Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin.J. Clin. Invest. 1999; 103: 613-625Crossref PubMed Scopus (129) Google Scholar). Moreover, lipopolysaccharide (LPS)-induced expression of E-selectin was inhibited by OxPAPC via cAMP and protein kinase A (PKA) (12Leitinger 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 (229) Google Scholar). Activation of α5β1 integrins and monocyte-endothelium adhesion by OxPAPC and POVPC was shown to be dependent on cAMP and R-Ras activation (13Cole A.L. Subbanagounder G. Mukhopadhyay S. Berliner J.A. Vora D.K. Oxidized phospholipid-induced endothelial cell/monocyte interaction is mediated by a cAMP-dependent R-Ras/PI3-kinase pathway.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1384-1390Crossref PubMed Scopus (123) Google Scholar). β1 integrins are rapidly activated by oxidized phospholipids within 15 min (11Shih P.T. Elices M.J. Fang Z.T. Ugarova T.P. Strahl D. Territo M.C. Frank J.S. Kovach N.L. Cabanas C. Berliner J.A. et al.Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin.J. Clin. Invest. 1999; 103: 613-625Crossref PubMed Scopus (129) Google Scholar). However, maximal β1 integrin activation and monocyte adhesion are observed after 4 h and require new protein synthesis (11Shih P.T. Elices M.J. Fang Z.T. Ugarova T.P. Strahl D. Territo M.C. Frank J.S. Kovach N.L. Cabanas C. Berliner J.A. et al.Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin.J. Clin. Invest. 1999; 103: 613-625Crossref PubMed Scopus (129) Google Scholar). Lipoxygenase activity and related arachidonate metabolites were shown to be necessary for MM-LDL to increase monocyte binding to HAECs (14Honda H.M. Leitinger N. Frankel M. Goldhaber J.I. Natarajan R. Nadler J.L. Weiss J.N. Berliner J.A. Induction of monocyte binding to endothelial cells by MM-LDL: role of lipoxygenase metabolites.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 680-686Crossref PubMed Scopus (76) Google Scholar). Recently, we demonstrated that OxPAPC induces tissue factor expression in human umbilical vein endothelial cells (HUVECs) by activating signaling pathways involving protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK 1/2), mitogen-activated protein kinase (MAPK), and increased intracellular calcium, ultimately activating the transcription factors endothelial growth related-1 (EGR-1) and nuclear factor of activated T-cells (NFAT) (9Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Oxidized phospholipids stimulate tissue factor expression in human endothelial cells via activation of ERK/EGR-1 and Ca(++)/NFAT.Blood. 2002; 99: 199-206Crossref PubMed Scopus (180) Google Scholar). Induction of interleukin 8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) by OxPAPC in endothelial cells has been suggested to involve peroxisome proliferator-activated receptor α (PPARα) (15Lee H. Shi W. Tontonoz P. Wang S. Subbanagounder G. Hedrick C.C. Hama S. Borromeo C. Evans R.M. Berliner J.A. et al.Role for peroxisome proliferator-activated receptor alpha in oxidized phospholipid-induced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells.Circ. Res. 2000; 87: 516-521Crossref PubMed Scopus (273) Google Scholar). In addition, OxPAPC-induced endothelial IL-8 expression was independent of the NF-κB signaling pathway (10Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar) and involved the activation of c-Src and signal transducer and activator of transcription 3 (STAT3) (10Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar). Activation of this pathway was mainly induced by 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine, which is another component phospholipid of OxPAPC (10Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar). In this study, we examined the signaling pathways induced by OxPAPC and POVPC that result in the specific adhesion of monocytes to HUVECs. We report that multiple signaling pathways involving PKA, PKC, ERK 1/2, and p38 MAPK need to be activated, resulting in the activation of cytosolic phospholipase A2(cPLA2) and the release of free arachidonic acid that is subsequently used by lipoxygenase, producing 12-hydroxyeicosatetraenoic acid (12-HETE), which acts as a signal mediator. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, DMSO, butylated hydroxytoluene, medium 199 (M199), and O-phenylene diamine were purchased from Sigma (St. Louis, MO). A multi-test Limulus amoebocyte lysate was from BioWhittaker (Walkersville, MD). The mitogen-activated ERK kinase (MEK) 1/2 inhibitor (PD098059), p38 inhibitor (SB203580), and PKC inhibitor (bisindolylmaleimide I) were obtained from Calbiochem (La Jolla, CA). 12(S)-HETE [12(S)-hydroxyeicosa-5Z, 8Z, 10E, 14Z-tetraenoic acid], cinnamoyl-3,4-dihydroxy-a-cyanocinnamate (CDC), and the cPLA2 inhibitor arachidonyltrifluoromethyl ketone were from Biomol; 5,8,11,14-eicosatetraynoic acid (ETYA) was from Cayman Chemical; arachidonic acid [5,6,8,9,11,12,14,15-3H(N)] was from Perkin-Elmer (Boston, MA). Tumor necrosis factor-α was from Boehringer Mannheim (Germany); anti-human E-selectin, ICAM-1, and VCAM-1 antibodies (all IgG) were from R&D Systems (Minneapolis, MN). Peroxidase-conjugated secondary anti-rabbit and anti-mouse antibodies were from Amersham Pharmacia Biotech (Uppsala, Sweden). Polyclonal antibodies against nonphosphorylated and phosphorylated ERK1/2 and LumiGLO were purchased from New England Biolabs (Beverly, MA). U937 cells and HL-60 cells were from the American Type Culture Collection (Manassas, VA), and supplemented calf serum was from HyClone (Logan, UT). HUVECs were prepared and cultured as described previously (16Zhang J.C. Fabry A. Paucz L. Wojta J. Binder B.R. Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells.Blood. 1996; 88: 3880-3886Crossref PubMed Google Scholar). Cells were used for experiments at passages three to five. OxPAPC and POVPC were prepared as described previously (4Watson 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 (692) Google Scholar, 5Subbanagounder 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 (199) Google Scholar). Lipids were tested for endotoxin content by Limulus test (from BioWhittaker). Only preparations containing <0.03 U/ml endotoxin were used for experiments. Adhesion assays were performed as described elsewhere (2Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions.J. Clin. Invest. 1990; 85: 1260-1266Crossref PubMed Scopus (769) Google Scholar, 17Leitinger N. Huber J. Rizza C. Mechtcheriakova D. Bochkov V. Koshelnick Y. Berliner J.A. Binder B.R. The isoprostane 8-iso-PGF(2alpha) stimulates endothelial cells to bind monocytes: differences from thromboxane-mediated endothelial activation.FASEB J. 2001; 15: 1254-1256Crossref PubMed Scopus (57) Google Scholar). In a previous publication (8Huber J. Boechzelt H. Karten B. Surboeck M. Bochkov V.N. Binder B.R. Sattler W. Leitinger N. Oxidized cholesteryl linoleates stimulate endothelial cells to bind monocytes via the extracellular signal-regulated kinase 1/2 pathway.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 581-586Crossref PubMed Scopus (43) Google Scholar), we demonstrated the similar behavior of monocyte-like U937 cells with respect to freshly isolated human peripheral blood mononuclear cells in adhesion assays. Briefly, confluent HUVECs on 48-well plates were incubated with agonists in M199 containing 10% supplemented calf serum for 4 h at 37°C. In some experiments, HUVECs were preincubated in the presence or absence of inhibitors dissolved in ethanol, DMSO, or solvent only, and then lipids dissolved in M199 or M199 only (control) were added for another 4 h. After incubation, HUVECs were washed and a suspension of unstimulated leukocytes (2 × 105 cells/well) was added for 15 min. Nonadherent leukocytes were removed by washing, and adherent cells were counted using an inverted phase-contrast microscope. Mouse aortic endothelial cells isolated from normal and PPARα null C57BL/6 mice were cultured as described previously (18Shi W. Wang N.J. Shih D.M. Sun V.Z. Wang X. Lusis A.J. Determinants of atherosclerosis susceptibility in the C3H and C57BL/6 mouse model: evidence for involvement of endothelial cells but not blood cells or cholesterol metabolism.Circ. Res. 2000; 86: 1078-1084Crossref PubMed Scopus (124) Google Scholar). After confluence in 48-well dishes, mouse aortic endothelial cells were stimulated with OxPAPC, LPS, or medium alone for 4 h at 37°C. After incubation, a suspension of unstimulated WEHI 78/24 cells was added for 15 min (19Tsao P.S. McEvoy L.M. Drexler H. Butcher E.C. Cooke J.P. Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine.Circulation. 1994; 89: 2176-2182Crossref PubMed Scopus (301) Google Scholar). Nonadherent leukocytes were removed by washing, and adherent cells were counted using an inverted phase-contrast microscope. After stimulation, HUVECs were lysed in Laemmli buffer and proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel. Proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), blocked with 5% dry milk/0.1% Tween-20, and incubated with primary antibodies in the same solution. Peroxidase-conjugated anti-IgG was used as a secondary antibody and detected by chemiluminescence (LumiGLO). Confluent HUVECs on 96-well plates were treated with agonists for 4 h at 37°C. The assay was then performed as described previously (20Krebs M. Kaun C. Lorenz M. Haag-Weber M. Geiger M. Binder B.R. Protease dependent activation of endothelial cells by peritoneal dialysis effluents.Thromb. Haemost. 1999; 82: 1334-1341Crossref PubMed Scopus (8) Google Scholar). In all leukocyte adhesion assays and ELISA experiments, tumor necrosis factor-α (20–50 U/ml) or LPS (2 ng/ml) was used as a positive control and yielded a reproducible 3- to 5-fold increase compared with cells treated with medium alone or containing a solvent vehicle, which is indicated as control. HUVECs were radiolabeled with 1 μCi/ml [3H]arachidonate culture M199 containing 10% fetal calf serum as described previously (21Tran K. Wong J.T. Lee E. Chan A.C. Choy P.C. Vitamin E potentiates arachidonate release and phospholipase A2 activity in rat heart myoblastic cells.Biochem. J. 1996; 319: 385-391Crossref PubMed Scopus (41) Google Scholar). After labeling, HUVECs were washed with HEPES-buffered saline containing 0.025% essentially fatty acid-free BSA and then stimulated with OxPAPC (150 μg/ml) or incubated with buffer alone (control) for the indicated time periods. The arachidonate released from cells was determined as described previously (21Tran K. Wong J.T. Lee E. Chan A.C. Choy P.C. Vitamin E potentiates arachidonate release and phospholipase A2 activity in rat heart myoblastic cells.Biochem. J. 1996; 319: 385-391Crossref PubMed Scopus (41) Google Scholar). Briefly, the supernatant was first collected and acetified with glacial acetic acid. Then, after lipid extraction, unlabeled arachidonic acid was added to the extraction mixture as a fatty acid carrier. The free fatty acid fraction was resolved by TLC with a solvent system consisting of hexane-diethyl ether-acetic acid (70:30:1, v/v) and was visualized by iodine vapor. Radioactivity was determined by liquid scintillation counting. A hammerhead ribozyme was designed to cleave the porcine leukocyte-type 12-lipoxygenase (12-LOX) RNA. The structure of the 12-LOX-hammerhead ribozyme has been described previously (22Gu J.L. Veerapanane D. Rossi J. Natarajan R. Thomas L. Nadler J. Ribozyme-mediated inhibition of expression of leukocyte-type 12-lipoxygenase in porcine aortic vascular smooth muscle cells.Circ. Res. 1995; 77: 14-20Crossref PubMed Scopus (39) Google Scholar, 23Gu J.L. Pei H. Thomas L. Nadler J.L. Rossi J.J. Lanting L. Natarajan R. Ribozyme-mediated inhibition of rat leukocyte-type 12-lipoxygenase prevents intimal hyperplasia in balloon-injured rat carotid arteries.Circulation. 2001; 103: 1446-1452Crossref PubMed Scopus (60) Google Scholar, 24Gu J.L. Nadler J. Rossi J. Use of a hammerhead ribozyme with cationic liposomes to reduce leukocyte type 12-lipoxygenase expression in vascular smooth muscle.Mol. Cell. Biochem. 1997; 172: 47-57Crossref PubMed Scopus (12) Google Scholar). Transfections of porcine aortic endothelial cells with ribozyme and monocyte adhesion assays were performed according to Patricia and coworkers (25Patricia M.K. Natarajan R. Dooley A.N. Hernandez F. Gu J.L. Berliner J.A. Rossi J.J. Nadler J.L. Meidell R.S. Hedrick C.C. Adenoviral delivery of a leukocyte-type 12 lipoxygenase ribozyme inhibits effects of glucose and platelet-derived growth factor in vascular endothelial and smooth muscle cells.Circ. Res. 2001; 88: 659-665Crossref PubMed Scopus (46) Google Scholar). Results are expressed as means ± SEM. Statistical analysis was performed using one-way ANOVA. P < 0.05 was considered statistically significant. HUVECs were stimulated with OxPAPC or POVPC, and the binding of monocyte-like U937 cells was determined. OxPAPC and POVPC stimulated HUVECs to bind monocytes (Fig. 1A), which is in accordance with previously published data on HAECs (4Watson 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 (692) Google Scholar, 12Leitinger 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 (229) Google Scholar, 26Watson A.D. Navab M. Hama S.Y. Sevanian A. Prescott S.M. Stafforini D.M. McIntyre T.M. Du B.N. Fogelman A.M. Berliner J.A. Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein.J. Clin. Invest. 1995; 95: 774-782Crossref PubMed Google Scholar). In contrast, OxPAPC did not induce the binding of neutrophil-like HL-60 cells to endothelial cells (Fig. 1A). To confirm these findings for human peripheral blood cells, we isolated monocytes and neutrophils from human blood. Consistently, OxPAPC stimulated the binding of human mononuclear cells, but not neutrophils, to HUVECs (Fig. 1B). Based on these results, we used monocyte-like U937 cells for further adhesion experiments. We have previously shown that OxPAPC did not upregulate the inflammatory adhesion molecules E-selectin, VCAM-1, or ICAM-1 but in contrast upregulated CS-1 fibronectin surface expression in HAECs (12Leitinger 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 (229) Google Scholar). Accordingly, treatment of HUVECs for 5 and 8 h did not result in the upregulation of E-selectin, VCAM-1, or ICAM-1 (Fig. 1C). However, adhesion of monocytes to OxPAPC-treated HUVECs was mediated by CS-1 fibronectin, because an antibody directed against CS-1, but not an irrelevant IgM, inhibited monocyte binding (Fig. 1D). OxPAPC and POVPC were shown to increase endothelial cAMP levels (12Leitinger 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 (229) Google Scholar, 27Parhami F. Fang Z.T. Yang B. Fogelman A.M. Berliner J.A. Stimulation of Gs and inhibition of Gi protein functions by minimally oxidized LDL.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 2019-2024Crossref PubMed Scopus (48) Google Scholar), which contributes to the induction of monocyte-endothelium interactions (13Cole A.L. Subbanagounder G. Mukhopadhyay S. Berliner J.A. Vora D.K. Oxidized phospholipid-induced endothelial cell/monocyte interaction is mediated by a cAMP-dependent R-Ras/PI3-kinase pathway.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1384-1390Crossref PubMed Scopus (123) Google Scholar). Inhibition of PKA, which is downstream of cAMP, by H89 reduced monocyte adhesion induced by OxPAPC in a concentration-dependent manner (Fig. 1E). These results show that the effects of OxPAPC on HUVECs are similar and comparable to those on HAECs. We have shown previously that treatment of HUVECs with OxPAPC induced the phosphorylation of ERK 1/2 within 20 min of stimulation, which was sustained for up to 8 h (9Bochkov V.N. Mechtcheriakova D. Lucerna M. Huber J. Malli R. Graier W.F. Hofer E. Binder B.R. Leitinger N. Oxidized phospholipids stimulate tissue factor expression in human endothelial cells via activation of ERK/EGR-1 and Ca(++)/NFAT.Blood. 2002; 99: 199-206Crossref PubMed Scopus (180) Google Scholar). To determine whether POVPC, one component lipid of OxPAPC, contributes to this effect, HUVECs were stimulated for 20 min with POVPC and phosphorylation of ERK 1/2 was detected by Western blotting. As shown in Fig. 2A, both OxPAPC and POVPC stimulated the phosphorylation of ERK 1/2 (Fig. 2A). Activation of ERK 1/2 requires phosphorylation by its upstream kinase MEK 1/2 (28Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions.Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1224) Google Scholar), which can be activated by a PKC-dependent pathway. To investigate the role of the PKC/MEK 1/2 and ERK 1/2 pathways in OxPAPC- and POVPC-induced endothelial-monocyte interactions, HUVECs were pretreated with a specific MEK 1/2 or PKC inhibitor (PD098059 or bisindolylmaleimide I, respectively) before addition of lipids. U937 cell binding induced by OxPAPC and POVPC was blocked by PD098059 (Fig. 2B, C) and bisindolylmaleimide I (Fig. 2D, E). Treatment of HUVECs with inhibitors alone or vehicle did not affect the basal level of adherent U937 cells (data not shown). Together, these results indicate that activation of HUVECs leading to monocyte-like U937 cell adhesion is dependent on PKC/MEK 1/2- and ERK 1/2-dependent pathways. In addition, we found that OxPAPC-induced U937 cell binding to HUVECs was blocked by the p38 inhibitor SB203580 (Fig. 3A). Addition of SB203580 or vehicle to untreated HUVECs had no effect on the level of U937 cell binding compared with control levels (data not shown). Moreover, phosphorylation of p38 was induced by OxPAPC (Fig. 3B). To examine whether the activation of p38 MAPK occurs independently from the PKC/ERK 1/2 pathway, HUVECs were incubated with specific inhibitors of PKC (bisindolylmaleimide I) and ERK 1/2 (PD098059) before stimulation with OxPAPC. Phosphorylation of p38 was determined subsequently by Western blot analysis. As shown in Fig. 3B, phosphorylation of p38 MAPK by OxPAPC was inhibited by neither bisindolylmaleimide I nor PD098059, whereas the p38 inhibitor SB203580 abolished the OxPAPC-induced phosphorylation of p38. Among the targets that are phosphorylated directly and thereby activated by ERK 1/2 and p38 MAPK is cPLA2 (29Sa G. Murugesan G. Jaye M. Ivashchenko Y. Fox P.L. Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells.J. Biol. 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