1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine and Inflammation

The generation of oxidized phospholipids in lipoproteins has been linked to vascular inflammation in atherosclerotic lesions. Products of phospholipid oxidation increase endothelial activation; however, their effects on macrophages are poorly understood, and it is unclear whether these effects are regulated by the biochemical pathways that metabolize oxidized phospholipids. We found that incubation of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) with THP-1-derived macrophages upregulated the expression of cytokine genes, including granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-1β, IL-6, and IL-8. In these cells, reagent POVPC was either hydrolyzed to lyso-phosphatidylcholine (lyso-PC) or reduced to 1-palmitoyl-2-(5-hydroxy-valeroyl)-sn-glycero-3-phosphocholine (PHVPC). Treatment with the phospholipase A(2) (PLA(2)) inhibitor, pefabloc, decreased POVPC hydrolysis and increased PHVPC accumulation. Pefabloc also increased the induction of cytokine genes in POVPC-treated cells. In contrast, PHVPC accumulation and cytokine production were decreased upon treatment with the aldose reductase (AR) inhibitor, tolrestat. In comparison with POVPC, lyso-PC led to 2- to 3-fold greater and PHVPC 10- to 100-fold greater induction of cytokine genes. POVPC-induced cytokine gene induction was prevented in bone-marrow derived macrophages from AR-null mice. These results indicate that although hydrolysis is the major pathway of metabolism, reduction further increases the proinflammatory responses to POVPC. Thus, vascular inflammation in atherosclerotic lesions is likely to be regulated by metabolism of phospholipid aldehydes in macrophages.

Topics: Aldehyde Reductase; Animals; Cell Line; Cytokines; Humans; Inflammation; Macrophages; Mice; Oxidation-Reduction; Phospholipid Ethers; Up-Regulation

Oxidized phospholipids, including oxidation products of palmitoyl-arachidonyl-phosphatidyl choline (PAPC), are mediators of inflammation in endothelial cells (ECs) and known to induce several chemokines, including interleukin-8 (IL-8). In this study, we show that oxidized PAPC (OxPAPC), which accumulates in atherosclerotic lesions, paradoxically depletes endothelial cholesterol, causing caveolin-1 internalization from the plasma membrane to the endoplasmic reticulum and Golgi, and activates sterol regulatory element-binding protein (SREBP). Cholesterol loading reversed these effects. SREBP activation resulted in increased transcription of the low-density lipoprotein receptor, a target gene of SREBP. We also provide evidence that cholesterol depletion and SREBP activation are signals for OxPAPC induction of IL-8. Cholesterol depletion by methyl-beta-cyclodextrin induced IL-8 synthesis in a dose-dependent manner. Furthermore, cholesterol loading of ECs by either the cholesterol-cyclodextrin complex or caveolin-1 overexpression inhibited OxPAPC induction of IL-8. These observations suggest that changes in cholesterol level can modulate IL-8 synthesis in ECs. The OxPAPC induction of IL-8 was mediated through the increased binding of SREBP to the IL-8 promoter region, as revealed by mobility shift assays. Overexpression of either dominant-negative SREBP cleavage-activating protein or 25-hydroxycholesterol significantly suppressed the effect of OxPAPC on IL-8 transcription. A role for SREBP activation in atherosclerosis is suggested by the observation that EC nuclei showed strong SREBP staining in human atherosclerotic lesions. The current studies suggest a novel role for endothelial cholesterol depletion and subsequent SREBP activation in inflammatory processes in which phospholipid oxidation products accumulate.

Topics: Animals; Aorta; Arteriosclerosis; beta-Cyclodextrins; Cattle; Caveolin 1; Caveolins; CCAAT-Enhancer-Binding Proteins; Cell Compartmentation; Cell Membrane; Cell Nucleus; Cells, Cultured; Cholesterol; DNA-Binding Proteins; Endoplasmic Reticulum; Endothelial Cells; Endothelium, Vascular; Golgi Apparatus; HeLa Cells; Humans; Hydroxycholesterols; Inflammation; Interleukin-8; Intracellular Signaling Peptides and Proteins; Membrane Lipids; Membrane Proteins; Phosphatidylcholines; Phospholipid Ethers; Recombinant Fusion Proteins; STAT3 Transcription Factor; Sterol Regulatory Element Binding Protein 1; Sterol Regulatory Element Binding Protein 2; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection

Monocyte recruitment into the vessel wall plays an important role in atherogenesis. Polar lipid components of minimally modified/oxidized LDL were shown to activate endothelial cells to increase the synthesis of monocyte chemotactic factors and surface expression of adhesion molecules. We previously reported regulation of endothelial cell inflammatory functions by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and three component oxidized phospholipids, containing oxovaleroyl (POVPC), glutaroyl (PGPC) and epoxyisoprostane (PEIPC) groups at the sn-2 position of oxidized phospholipids. In the present study, we demonstrate the presence of gamma-hydroxy-alpha,beta-unsaturated aldehydic phospholipid, 1-palmitoyl-2-(5-hydroxy-8-oxooct-6-enoyl)-sn-glycero-3-phosphocholine (HOOA-PC; m/z 650.4), in Ox-PAPC by liquid chromatography/mass spectrometry (LC/MS), LC/MS/MS, derivatization and tandem mass spectrometric analyses. This was further unambiguously confirmed by the identical chromatographic and mass spectrometric characteristics of Ox-PAPC-derived m/z 650.4 with synthetic HOOA-PC. The time course of PAPC autoxidation showed that HOOA-PC accumulates with oxidation and represents about 2% of Ox-PAPC. We have also examined the effects of HOOA-PC on leukocyte-endothelial interactions. HOOA-PC dose-dependently activated human aortic endothelial cells (HAECs) to bind monocytes (twofold at 10 micrograms/ml) and caused a dose-dependent increase (two- to threefold) in levels of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8)--chemokines that are important in monocyte entry into chronic lesions. HOOA-PC also inhibited LPS-induced expression of E-Selectin, a major adhesion molecule that mediates neutrophil endothelial interactions. The present study suggests that the HOOA-PC exerts its effects on endothelial cells as a free lipid. These studies demonstrate the importance of HOOA-PC as a new potential proinflammatory molecule that regulates leukocyte-endothelial interactions.

Topics: Anti-Inflammatory Agents; Cells, Cultured; Chemokines; Chromatography, High Pressure Liquid; E-Selectin; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Glycerylphosphorylcholine; Humans; Inflammation; Lipopolysaccharides; Mass Spectrometry; Oleic Acids; Oxidation-Reduction; Phospholipid Ethers; Phospholipids; Spectrometry, Mass, Electrospray Ionization; Time Factors