1-o-hexadecyl-2-arachidonyl-sn-glycero-3-phosphocholine and sodium-borohydride

We previously described 3 bioactive oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) containing oxovaleroyl (POVPC), glutaroyl (PGPC), and epoxyisoprostane (PEIPC) groups at the sn-2 position that were increased in minimally modified/oxidized low density lipoprotein (MM-LDL) and rabbit atherosclerotic lesions. We demonstrated specific and contrasting effects of POVPC and PGPC on leukocyte-endothelial interactions and described an effect of PEIPC on monocyte binding. The major purpose of the present study was to determine the effects of structural changes on the bioactivities of these 3 lipids. We demonstrate herein that the group at the sn-2 position determines the specific bioactivity and that the substitution of stearoyl for palmitoyl at the sn-1 position or ethanolamine for choline at the sn-3 position of the phospholipid did not alter bioactivity. Oxidized PAPC, oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, and oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylethanolamine stimulated monocyte binding and inhibited lipopolysaccharide-induced expression of the neutrophil-binding molecule E-selectin. Furthermore, all oxovaleroyl phospholipids but not the glutaroyl phospholipids induced monocyte binding without an increase in vascular cell adhesion molecule-1 (VCAM-1) expression and inhibited lipopolysaccharide-induced E-selectin expression. In contrast, glutaroyl phospholipids but not oxovaleroyl phospholipids stimulated E-selectin and VCAM-1 expression. We further demonstrate that all parts of the phospholipid molecules are required for these bioactivities. Hydrolysis with phospholipase (PL) A(1), PLA(2), and PLC strongly reduced the bioactivities of POVPC, PGPC, and mixed isomers of PEIPC. PLD had a smaller but still significant effect. The effects of POVPC and PEIPC could be abolished by sodium borohydride treatment, indicating the importance of the reducible groups (carbonyl and epoxide) in these molecules. In summary, these studies identify 6 new bioactive, oxidized phospholipids that are increased in MM-LDL and, where measured, in atherosclerotic lesions. They thus suggest that a family of phospholipid oxidation products containing oxovaleroyl, glutaroyl, and epoxyisoprostane at the sn-2 position play an important role in the regulation of leukocyte-endothelial interactions, bioactivity being in part controlled by several types of phospholipid hydrolases.

Topics: Animals; Aorta; Arteriosclerosis; Borohydrides; Diet, Atherogenic; E-Selectin; Lipopolysaccharides; Lipoproteins, LDL; Molecular Structure; Monocytes; Oxidation-Reduction; Phospholipases; Phospholipid Ethers; Rabbits; Stereoisomerism; Vascular Cell Adhesion Molecule-1

Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis.

Topics: Animals; Antibodies, Monoclonal; Arteriosclerosis; Borohydrides; Cell Adhesion; Chromatography, High Pressure Liquid; Endothelium, Vascular; Fluorobenzenes; Hydroxylamines; Lipoproteins, LDL; Mass Spectrometry; Mice; Monocytes; Oxidation-Reduction; Phospholipid Ethers; Phospholipids; Rabbits