Evidence for a role of phospholipid oxidation products in atherogenesis.

There is growing evidence for the accumulation of phospholipid oxidation products (some of which can also be formed enzymatically) in several chronic disease processes including atherosclerosis. There also is considerable evidence that enzymes involved in hydrolysis of these phospholipids (present in both lipoproteins and cells) may be important in regulation of atherogenesis. In vitro studies suggest that these lipids can activate vascular wall cells to states that contribute to the atherosclerotic process. This review focuses on two types of bioactive phospholipids: phosphatidyl cholines in which the sn-2 fatty acid has been modified by oxidation and lysophosphatidic acid in which both the sn-2 and sn-3 positions have been modified. The mechanism by which these phospholipid oxidation products activate cells has revealed the presence of several different receptors and signal transduction pathways.

A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.

We have developed a novel and rapid cell-free assay of the ability of HDL to prevent the formation of or inactivate oxidized phospholipids. HDL was tested for its ability to inhibit the oxidation of LDL, or inhibit the oxidation of l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) by hydroperoxyoctadecadienoic acid (HPODE), or inactivate oxidized PAPC (Ox-PAPC). In each case the fluorescent signal generated in the presence of the test substances and the test HDL was determined. As little as 2.5 microg of normal human HDL cholesterol significantly inhibited the fluorescent signal generated by Ox-PAPC; results did not differ regardless of whether the HDL was prepared by gel electrophoresis, fast protein liquid chromatography, or dextran sulfate precipitation. HDL from each of 27 patients with coronary atherosclerosis failed to inhibit the fluorescent signal generated by a control LDL, whereas HDL from each of 31 matched normal subjects with the same levels of HDL cholesterol significantly inhibited the signal. Results from an established cell-based assay (Navab, M., S. Hama, J. Cooke, G. M. Anantharamaiah, M. Chaddha, L. Jin, G. Subbanagounder, K. F. Faull, S. T. Reddy, N. E. Miller, and A. M. Fogelman. 2000. J. Lipid Res. 41: 1481-1494) were identical. HDL from the patients also failed to inhibit the fluorescent signal generated from PAPC plus HPODE (10 of 10 patients) whereas HDL from matched controls (8 of 8 patients) significantly inhibited the fluorescent signal. We conclude that this new assay has the potential to allow widespread testing of the hypothesis that HDL that is dysfunctional in preventing the formation or inactivating oxidized phospholipids may play an important role in the development of atherosclerosis.

HDL and the inflammatory response induced by LDL-derived oxidized phospholipids.

Oxidation of low density lipoprotein (LDL) phospholipids containing arachidonic acid at the sn-2 position occurs when a critical concentration of "seeding molecules" derived from the lipoxygenase pathway is reached in LDL. When this critical concentration is reached, the nonenzymatic oxidation of LDL phospholipids produces a series of biologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. Normal high density lipoprotein (HDL) contains at least 4 enzymes as well as apolipoproteins that can prevent the formation of the LDL-derived oxidized phospholipids or inactivate them after they are formed. In the sense that normal HDL can prevent the formation of or inactivate these inflammatory LDL-derived oxidized phospholipids, normal HDL is anti-inflammatory. HDL from mice that are genetically predisposed to diet-induced atherosclerosis became proinflammatory when the mice are fed an atherogenic diet, injected with LDL-derived oxidized phospholipids, or infected with influenza A virus. Mice that were genetically engineered to be hyperlipidemic on a chow diet and patients with coronary atherosclerosis, despite normal lipid levels, also had proinflammatory HDL. It is proposed that LDL-derived oxidized phospholipids and HDL may be part of a system of nonspecific innate immunity and that the detection of proinflammatory HDL may be a useful marker of susceptibility to atherosclerosis.

Determinants of bioactivity of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position.

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.

Role for peroxisome proliferator-activated receptor alpha in oxidized phospholipid-induced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells.

The attraction, binding, and entry of monocytes into the vessel wall play an important role in atherogenesis. We have previously shown that minimally oxidized/modified LDL (MM-LDL), a pathogenically relevant lipoprotein, can activate human aortic endothelial cells (HAECs) to produce monocyte chemotactic activators. In the present study, we demonstrate that MM-LDL and oxidation products of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) activate endothelial cells to synthesize monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8). Several lines of evidence suggest that this activation is mediated by the lipid-dependent transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha), the most abundant member of the PPAR family in HAECs. Treatment of transfected CV-1 cells demonstrated activation of the PPARalpha ligand-binding domain by MM-LDL, Ox-PAPC, or its component phospholipids, 1-palmitoyl-2-oxovalaroyl-sn-glycero-phosphocholine and 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine; these lipids also activated a consensus peroxisome proliferator-activated receptor response element (PPRE) in transfected HAECs. Furthermore, activation of PPARalpha with synthetic ligand Wy14,643 stimulates the synthesis of IL-8 and MCP-1 by HAECs. By contrast, troglitazone, a PPARgamma agonist, decreased the levels of IL-8 and MCP-1. Finally, we demonstrate that unlike wild-type endothelial cells, endothelial cells derived from PPARalpha null mice do not produce MCP-1/JE in response to Ox-PAPC and MM-LDL. Together, these data demonstrate a proinflammatory role for PPARalpha in mediation of the activation of endothelial cells to produce monocyte chemotactic activity in response to oxidized phospholipids and lipoproteins.

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1.

Apolipoprotein A-I (apoA-I) and an apoA-I peptide mimetic removed seeding molecules from human low density lipoprotein (LDL) and rendered the LDL resistant to oxidation by human artery wall cells. The apoA-I-associated seeding molecules included hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE). LDL from mice genetically susceptible to fatty streak lesion formation was highly susceptible to oxidation by artery wall cells and was rendered resistant to oxidation after incubation with apoA-I in vitro. Injection of apoA-I (but not apoA-II or murine serum albumin) into mice rendered their LDL resistant to oxidation within 3 h. Infusion of apoA-I into humans rendered their LDL resistant to oxidation within 6 h. We conclude that 1) oxidation of LDL by artery wall cells requires seeding molecules that include HPODE and HPETE; 2) LDL from mice genetically susceptible to atherogenesis is more readily oxidized by artery wall cells; and 3) normal HDL and its components can remove or inhibit the activity of lipids in freshly isolated LDL that are required for oxidation by human artery wall cells.

Bioactive products of phospholipid oxidation: isolation, identification, measurement and activities.

There is considerable evidence to suggest that oxidation of LDL plays an important role in atherogenesis. Polyunsaturated fatty acids, a major oxidative target, are present as phospholipids in the outer core of the lipoprotein particle. Studies from several laboratories have shown an increase in the levels of phospholipid oxidation products in atherosclerotic lesions and of antibodies to oxidized phospholipids in mice and humans with lesions. Significantly, phospholipid oxidation products have been demonstrated (in vitro) to selectively activate processes in vascular wall cells that may contribute to atherogenesis. This review discusses activities, methods for isolation, identification and measurement of bioactive phospholipids. Past studies suggest that defined and relatively simple current technologies allow identification of bioactive phospholipid oxidation products and measurement of their levels in tissue.

Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis.

Serum paraoxonase (PON1), present on high density lipoprotein, may inhibit low density lipoprotein (LDL) oxidation and protect against atherosclerosis. We generated combined PON1 knockout (KO)/apolipoprotein E (apoE) KO and apoE KO control mice to compare atherogenesis and lipoprotein oxidation. Early lesions were examined in 3-month-old mice fed a chow diet, and advanced lesions were examined in 6-month-old mice fed a high fat diet. In both cases, the PON1 KO/apoE KO mice exhibited significantly more atherosclerosis (50-71% increase) than controls. We examined LDL oxidation and clearance in vivo by injecting human LDL into the mice and following its turnover. LDL clearance was faster in the double KO mice as compared with controls. There was a greater rate of accumulation of oxidized phospholipid epitopes and a greater accumulation of LDL-immunoglobulin complexes in the double KO mice than in controls. Furthermore, the amounts of three bioactive oxidized phospholipids were elevated in the endogenous intermediate density lipoprotein/LDL of double KO mice as compared with the controls. Finally, the expression of heme oxygenase-1, peroxisome proliferator-activated receptor gamma, and oxidized LDL receptors were elevated in the livers of double KO mice as compared with the controls. These data demonstrate that PON1 deficiency promotes LDL oxidation and atherogenesis in apoE KO mice.

Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils.

We previously have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified low density lipoprotein (MM-LDL), activates endothelial cells to bind monocytes. 1-Palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), which are present in OxPAPC, MM-LDL, and atherosclerotic lesions, were shown to have a major role in the activation of endothelial cells. We now demonstrate that these two highly similar molecules have dramatically different effects on leukocyte endothelial interactions. POVPC is a potent regulator of monocyte-specific endothelial interactions. Treatment of endothelial cells with POVPC increased monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin; no increase in neutrophil binding was observed. In addition, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition was mediated by a protein kinase A-dependent pathway, resulting in down-regulation of NF-kappaB-dependent transcription. In contrast, PGPC induced both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule 1. We present evidence to suggest that the two phospholipids act by different novel receptors present in Xenopus laevis oocytes and that POVPC, but not PGPC, stimulates a cAMP-mediated pathway. At concentrations equal to that present in MM-LDL, the effect of POVPC dominates and inhibits PGPC-induced neutrophil binding and E-selectin expression in endothelial cells. In summary, our data provide evidence that both POVPC and PGPC are important regulators of leukocyte-endothelial interactions and that POVPC may play a dominant role in a number of chronic inflammatory processes where oxidized phospholipids are known to be present.

Evidence that phospholipid oxidation products and/or platelet-activating factor play an important role in early atherogenesis : in vitro and In vivo inhibition by WEB 2086.

The goal of the present studies was to determine whether phospholipid oxidation products and/or platelet-activating factor (PAF) are mediators of early atherogenesis in vivo. Monocyte-endothelial cell interactions have been shown to play an important role in early atherogenesis. We and others have demonstrated that PAF and phospholipid oxidation products, present in atherosclerotic lesions, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-epoxyisoprostane E(2)-sn-glycero-3-phosphocholine (PEIPC), mediate the activation of monocytes and/or endothelial cells in vitro. Previous studies have shown that the action of PAF and PAF-like ether-containing phospholipids was inhibited by WEB 2086. We now demonstrate that pretreatment of human aortic endothelial cells with WEB 2086 (10 micromol/L) and several other PAF antagonists before treatment with POVPC and PEIPC but not PGPC prevented the activation of the endothelial cells to bind monocytes. We present evidence to suggest that this inhibition is not mediated by the PAF receptor. The role of bioactive oxidized phospholipids in fatty streak formation was tested using C57BL/6J LDL R-/- mice fed a chow or Western diet for 5 weeks with or without WEB 2086 mixed with drinking water. Quantitative electrospray ionization mass spectrometry showed similar concentrations of WEB 2086 in the plasma of mice on both diets ( approximately 4 to 5 micromol/L); this concentration was inhibitory in vitro. Administration of WEB 2086 did not affect the lipid composition of mouse plasma. However, fatty streak formation was reduced by 62% in animals fed a Western diet, whereas no change was observed in the small lesions of mice on a chow diet. These studies provide evidence that PAF and/or PAF-like phospholipid oxidation products are important mediators of atherosclerotic lesion development in vivo and that specific receptor antagonists for these molecules may represent a novel therapeutic modality.

Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.

One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A(2) and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5, 6-epoxyisoprostane E(2))-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.

Protein adducts of iso[4]levuglandin E2, a product of the isoprostane pathway, in oxidized low density lipoprotein.

Levuglandin (LG) E2, a cytotoxic seco prostanoic acid co-generated with prostaglandins by nonenzymatic rearrangements of the cyclooxygenase-derived endoperoxide, prostaglandin H2, avidly binds to proteins. That LGE2-protein adducts can also be generated nonenzymatically is demonstrated by their production during free radical-induced oxidation of low density lipoprotein (LDL). Like oxidized LDL, LGE2-LDL, but not native LDL, undergoes receptor-mediated uptake and impaired processing by macrophage cells. Since radical-induced lipid oxidation produces isomers of prostaglandins, isoprostanes (isoPs), via endoperoxide intermediates, we postulated previously that a similar family of LG isomers, isoLGs, is cogenerated with isoPs. Now iso[4]LGE2-protein epitopes produced by radical-induced oxidation of arachidonic acid in the presence of protein were detected with an enzyme-linked immunosorbent assay. Iso[4]LGE2-protein epitopes are also generated during free radical-induced oxidation of LDL. All of the LGE2 isomers generated upon oxidation of LDL are efficiently sequestered by covalent adduction with LDL-based amino groups. The potent electrophilic reactivity of iso-LGs can be anticipated to have biological consequences beyond their obvious potential as markers for specific arachidonate-derived protein modifications that may be of value for the quantitative assessment of oxidative injury.

Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid-protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins.

We recently cloned monoclonal IgM autoantibodies which bind to epitopes of oxidized low-density lipoprotein (OxLDL) from apoE-deficient mice (EO- autoantibodies). We now demonstrate that those EO- autoantibodies that were originally selected for binding to copper-oxidized low-density lipoproteins (CuOx-LDL), also bound both to the oxidized protein and to the oxidized lipid moieties of CuOx-LDL. The same EO- autoantibodies showed specific binding to products of oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine (OxPAPC) and to the specific oxidized phospholipid, 1-palmitoyl-2-(5-oxovaleroyl)-phosphatidyl-choline (POVPC), whereas oxidation of fatty acids (linoleic or arachidonic acid) or cholesteryl esters (cholesteryl-oleate or cholesteryl-linoleate) did not yield any binding activity. Those EO- autoantibodies that bound to oxidized phospholipids (e.g., EO6) inhibited the binding and degradation of CuOx-LDL by mouse peritoneal macrophages up to 91%, whereas other IgM EO- autoantibodies, selected for binding to malondialdehyde (MDA)-LDL, had no influence on binding of either CuOx-LDL or MDA-LDL by macrophages. F(ab')2 fragments of EO6 were equally effective as the intact EO6 in preventing the binding of CuOx-LDL by macrophages. The molar ratios of IgM to LDL needed to maximally inhibit the binding varied from approximately 8 to 25 with different CuOx-LDL preparations. Finally, a POVPC-bovine serum albumin (BSA) adduct also inhibited CuOx-LDL uptake by macrophages. These data suggest that oxidized phospholipid epitopes, present either as lipids or as lipid-protein adducts, represent one class of ligands involved in the recognition of OxLDL by macrophages, and that apoE-deficient mice have IgM autoantibodies that can bind to these neoepitopes and inhibit OxLDL uptake.

Oxidation of low-density lipoproteins produces levuglandin-protein adducts.

Free-radical oxidation of human low-density lipoprotein (LDL) produces levuglandin (LG)-protein adducts that were detected with an enzyme-linked immunosorbent assay using LGE2-KLH antibodies which recognize LGE2-derived pyrroles. The level of immunoreactivity increases with time of oxidation and reaches a maximum by 8 h. The yield of pyrrole varies nonlinearly with the level of LG adduction to LDL. At low LG:LDL ratios, such as those detected in oxidized LDL, the reaction of primary amino groups with LGE2 produces mostly non-pyrrole adducts that are not immunoreactive. Concomitant phospholipolysis must occur if the generation of immunoreactive epitopes in LDL involves oxidation of arachidonyl phospholipids. Thus, since a protein adduct prepared from synthetic LGE2-2-lysophosphatidylcholine ester showed, at most, only 0.5% cross-reactivity with the LGE2-KLH antibodies, the epitopes detected in oxidized LDL are almost certainly not protein adducts of LG-phospholipid esters. As expected, hydrolysis of the carboxylic ester in the protein adduct of LGE2-2-lysophosphatidylcholine ester by treatment with phospholipase A2 produced a fully immunoreactive LGE2-protein adduct.

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.

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.

Levuglandin E2-protein adducts in human plasma and vasculature.

The prostaglandin endoperoxide PGH2 rearranges nonenzymatically to generate prostaglandins and secoprostanoic acid levulinaldehyde derivatives such as PGE2 and levuglandin (LG) E2, respectively. Direct detection of LGE2 in biological samples is complicated because it is rapidly sequestered by covalent adduction to endogenous nucleophiles including proteins, which produces LGE2-derived protein-bound pyrroles. Therefore, to detect LGE2-protein adducts in vivo, antibodies were raised against a covalent adduct of LGE2 with keyhole limpet hemocyanin (KLH). This antigen enabled the production of high-titer antibodies that exhibit minimal cross-specificity and are sensitive for detecting LGE2-derived pyrroles. Although pyrrole yields are low at LG/protein ratios found in vivo, an enzyme-linked immunosorbent assay with the LGE2-KLH antibodies detects LGE2-derived protein-bound pyrrole immunoreactivity in human plasma from specific patient populations. Furthermore, prominent immunocytochemical staining of human brain thin sections revealed the presence of LGE2-derived pyrrole immunoreactivity, especially in the meningeal vessels of some patients. This demonstration of LG-protein adducts in human plasma and vasculature provides the first evidence for the biological occurrence of levuglandins in vivo and further suggests that these antibodies might prove useful in diagnostic and mechanistic studies of various disease conditions.

Macrophage recognition of LDL modified by levuglandin E2, an oxidation product of arachidonic acid.

Levuglandin (LG) E2, a secoprostanoic acid levulinaldehyde derivative, is a product of free radical oxidation that forms covalent adducts with lysyl residues on proteins. Treatment of LDL with LGE2 leads to uptake and degradation by mouse peritoneal macrophages. Oxidized LDL, but not acetyl LDL efficiently competed for binding and uptake of LGE2-modified 125I-LDL. This result suggests that LGE2-modified LDL was recognized by a class of scavenger receptor that demonstrated ligand specificity for oxidized LDL but not for acetyl LDL.

Immunochemical evidence supporting 2-pentylpyrrole formation on proteins exposed to 4-hydroxy-2-nonenal.

Previous model studies suggested the formation of lysine-based 2-pentylpyrroles as novel late adduction products formed upon exposure of proteins to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Two 2-pentylpyrrole immunogens were prepared, one by treating keyhole limpet hemocyanin (KLH) directly with 4-oxononanal and the other by preformation of 6-(2-pentylpyrrol-1-yl)hexanoic acid from 6-aminocaproic acid and 4-oxononanal, followed by carbodiimide coupling to KLH. Pyrrole content and lysine modification in KLH were assayed independently. Following immunization of rabbits, antibody titer increased and plateaued over a 4 month period. The structural specificity of the IgG fractions of the antisera was evaluated through comprehensive competitive ELISA studies. These antibodies were used to verify the time-dependent appearance of the 2-pentylpyrrole epitope in protein exposed to HNE. Potential advantages of antibodies recognizing "advanced lipid peroxidation end products" over those recognizing "early" HNE adduction products are discussed.

Formation and stability of pyrrole adducts in the reaction of levuglandin E2 with proteins.

Levuglandin (LG) E2 is rapidly sequestered by covalent binding with proteins. The reaction of LGE2 with a protein in neutral aqueous solution exhibits two phases. A metastable adduct rapidly accumulates initially. In the second phase, a protein-bound pyrrole is generated. Pyrrole formation and stability were monitored with an immunoassay using antibodies that were raised against a stable isostere. That LG-derived pyrroles are the major products (> 76%) of the LGE2-protein reaction is suggested by the level of antibody binding found for LG-protein adducts compared with that found for a pyrrole derived from LGE2 and 6-amino-1-hexanol. Because the initial metastable LG-protein adduct is a reactive electrophile, it can be trapped with amines, such as glycine, to give stable ternary adducts that do not cross-react with the antibodies. Although highly alkylated pyrroles are chemically sensitive compounds, the protein-bound LG-derived pyrrole appears to be stable in aqueous solution at pH 7.4. Thus, it shows no decrease in immunoreactivity over several weeks. This discovery leads to the expectation that such pyrroles will accumulate in vivo, especially in proteins that do not turn over rapidly. Thus, the LG-derived protein-bound pyrrole may be a useful marker of oxidative lipid damage, and an immunoassay for this post-translational protein modification can be exploited as a mild, sensitive method for detecting and quantifying the generation of LGs in chronic inflammatory states.