Abstract
Monocyte recruitment to the vessel wall, mediated by monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8), plays an important role in atherogenesis. We have shown previously that minimally oxidized low density lipoprotein, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), activates endothelial cells to produce MCP-1 and IL-8. By using liquid chromatography/mass spectrometry methods coupled with bioassay, we report a family of epoxyisoprostane (PEIPC) and epoxycyclopentenone (PECPC) phospholipids that are the components of Ox-PAPC responsible for the majority of this activity. Ox-PAPC contains five chromatographically distinguishable active PEIPC components (m/z 825.5) and four PECPC components (m/z 810.5). All nine components induced endothelial cell synthesis of IL-8 and MCP-1 in a dose-dependent fashion between 0.1 and 5 microm concentrations. The five PEIPC components had identical functional groups and all underwent dehydration to produce m/z 810.5. We present evidence that these phospholipids are regioisomers with epoxide groups at the 5,6-, 8,9-, 11,12-, or 14,15-positions of the sn-2 fatty acid and their epoxide groups is important for biological activity. We have shown previously that peroxisome proliferator-activated receptor alpha is involved in MCP-1 synthesis in response to Ox-PAPC. We now show that PEIPC and PECPC isomers are potent activators of peroxisome proliferator-activated receptor alpha. PEIPC and PECPC isomers are strongly recognized by specific circulating murine natural autoantibodies (EO6) and accumulate in cells treated with IL-1beta. These studies demonstrate that PEIPC and PECPC isomers are potent activators of endothelial cells increasing synthesis of IL-8 and MCP-1. Their accumulation in cells exposed to cytokines and in atherosclerotic lesions suggests that these lipids may play a role in a number of chronic disease processes.
Highlights
Monocyte recruitment to the vessel wall, mediated by monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8), plays an important role in atherogenesis
By using liquid chromatography/mass spectrometry methods coupled with bioassay, we report a family of epoxyisoprostane (PEIPC) and epoxycyclopentenone (PECPC) phospholipids that are the components of oxidized 1-palmitoyl-2-arachidonylsn-glycero-3-phosphorylcholine (Ox-PAPC) responsible for the majority of this activity
We have identified a family of PEIPC and PEIPC and the corresponding dehydration products (PECPC) derivatives that were more potent inducers of chemokine production than the two previously identified biologically active phospholipids present in minimally modified/oxidized LDL (MM-LDL) (POVPC and PGPC)
Summary
Ox-PAPC, oxidized 1-palmitoyl-2arachidonyl-sn-glycero-3-phosphorylcholine; MCP-1, monocyte chemotactic protein-1; IL-8, interleukin-8; LDL, low density lipoprotein; MMLDL, minimally modified/oxidized LDL; OxLDL, oxidized LDL; LC/MS, liquid chromatography/mass spectrometry; RP, reverse phase; NP, normal phase; HPLC, high pressure liquid chromatography; PPAR, peroxisome proliferator-activated receptor; PEIPC, 1-palmitoyl-2-(epoxyisoprostane)-sn-glycero-3-phosphorylcholine; PECPC, 1-palmitoyl-2(epoxycyclopentenone)-sn-glycero-3-phosphorylcholine; POVPC, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine; PGPC, 1palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine; ANOVA, analysis of variance; PLA2, phospholipase A2; DMPC, dimyristoyl-sn-glycero3-phosphorylcholine; BHT, butylated hydroxytoluene; HAEC, human aortic endothelial cells; MRM, multiple reaction monitoring; PCA, perchloic acid; FBS, fetal bovine serum; LPDS, lipoprotein-deficient serum; PC, phosphorylcholine; ESIMS, electrospray ionization mass spectrometry; PBS, phosphate-buffered saline; BSTFA, bis(trimethylsilyl)trifluoroacetamide. Two components of MM-LDL and Ox-PAPC, POVPC and PGPC, increased IL-8 and MCP-1 but were much less active than Ox-PAPC suggesting there were additional active molecules present in OxPAPC These studies identify epoxyisoprostane and epoxycyclopentenone phospholipids as the major components of OxPAPC inducing MCP-1 and IL-8. The present studies demonstrate that epoxyisoprostane and epoxycyclopentenone phospholipids are potent activators of PPAR␣ Another goal of the current studies was to determine whether bioactive phospholipids regulating chemokine synthesis could be produced in cell membranes in response to agents known to increase free radical production. There is evidence that oxidized phospholipids may be present in chronic diseases other than atherosclerosis, and we hypothesized that these might arise from cell membrane oxidation To test this hypothesis, the ability of interleukin-1 (IL-1), an important regulator of many processes of chronic inflammation, to induce the formation of bioactive phospholipids in endothelial cells was examined
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