Abstract
Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiate atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA), also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells. γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in human umbilical cord endothelial cells. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal induced THP-1 adhesion, but N-glutaroyl-PE and C(18:0)N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and 4-oxo-pentanal significantly reduced the temperature for the liquid crystalline to hexagonal phase transition in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER); γKA-PE induced C/EBP homologous protein CHOP and BiP expression and p38 MAPK activity, and inhibitors of ER stress reduced γKA-PE-induced C/EBP homologous protein CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.
Highlights
Lipid peroxidation has been implicated in a host of pathological processes, including inflammation and atherosclerosis, but there is still much that is unknown about the mechanisms linking lipid peroxidation to the activation of inflammatory responses
PE Modified by an Endogenous ␥-Ketoaldehyde Induces Endothelial Cell Activation—ROS have previously been associated with activation of endothelial cells and inflammation
Our results demonstrate that PE modification is biologically important because PE modified by ␥KA activates the proinflammatory response of endothelial cells
Summary
Reagents—1,2-Dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DiPoPE), 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine (DPPE), 1-palmitoyl-2-hydroxysn-glycero-3-phosphoethanolamine (LPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (C16:0 glutaryl-PE (glt-PE)), and 1-palmitoyl-2-{12-[(7-nitro-21,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-glycero-3-phosphoethanolamine (NBD-PE) were purchased from Avanti Polar Lipids (Alabaster, AL). To assess the extent that each N-modified PE as incorporated by HUVEC, we incubated the mixture containing 1 M of each N-modified PE dissolved in the DMEM, 0.1% HSA, 0.5% ethanol with triplicate wells of HUVEC for 4 h, removed the mixture, washed the cells twice, added C17: 0NAPE as internal standard, extracted the cellular phospholipids with chloroform/methanol (2:1) solution, and analyzed by mass spectrometry. To determine whether ␥KA-NBD-PE remained intact or was hydrolyzed during the incubation with HUVEC prior to fluorescence imaging, we incubated ␥KA-NBD-PE with either cellfree buffer or with HUVEC for 1 h at 4 °C and for 15 min at 37 °C After this incubation period, the solution was removed from cells; lipids were extracted using acidic modified BlighDyer to ensure extraction of NBD-fatty acid metabolites, and the products were analyzed by TLC using chloroform, methanol, ethanol, ethyl acetate, 0.25% potassium chloride (10:4:10: 10:3.6) as solvent. Statistical Analysis—All statistical analysis was performed using Graph Pad Prism 4.03 (GraphPad Software, La Jolla, CA)
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