Structural identification and cardiovascular activities of oxidized phospholipids - PubMed (original) (raw)
Review
Structural identification and cardiovascular activities of oxidized phospholipids
Robert G Salomon. Circ Res. 2012.
Abstract
Free radical-induced oxidation of membrane phospholipids generates complex mixtures of oxidized phospholipids (oxPLs). The combinatorial operation of a few dozen reaction types on a few dozen phospholipid structures results in the production of a dauntingly vast diversity of oxPL molecular species. Structural identification of the individual oxPL in these mixtures is a redoubtable challenge that is absolutely essential to allow determination of the biological activities of individual species. With an emphasis on cardiovascular consequences, this Review focuses on biological activities of oxPLs whose molecular structures are known and highlights 2 diametrically opposite approaches that were used to determine those structures, that is, (1) the classic approach from bioactivity of a complex mixture to isolation and structural characterization of the active molecule followed by confirmation of the structure by unambiguous chemical synthesis and (2) hypothesis of products that are likely to be generated by lipid oxidation, followed by synthesis, and then detection in vivo guided by the availability of authentic standards, and last, characterization of biological activities. Especially important for the application of the second paradigm is the capability of LC-MS/MS and derivatizations to selectively detect and quantify specific oxPL in complex mixtures, without the need for their isolation or complete separation. This technology can provide strong evidence for identity by comparisons with pure, well-characterized samples available by chemical syntheses. Those pure samples are critical for determining the biological activities attributable to specific molecular species of oxPLs in the complex mixtures generated in vivo as a consequence of oxidative stress.
Figures
Figure 1. Polyunsaturated phospholipid peroxyl radicals undergo cyclization and fragmentation reactions
Free radical-initiated oxidation of polyunsaturated phospholipids (PL = 2-lyso phospholipid) by hydrogen atom abstraction from doubly allylic CH2 groups generates pentadienyl radicals that react with molecular oxygen to produce peroxyl radicals. Multifunctional cyclic oxPLs are generated through cyclization of peroxyl radicals and reaction with additional molecular oxygen can produce hydroperoxy endoperoxides that rearrange to hydroperoxy isoprostanes. Dehydrative cyclization delivers epoxy isoprostane phospholipids, and further dehydration produces epoxycyclopentenone PLs. Oxidative fragmentation of peroxyl radicals produces oxidatively truncated oxPLs. γ-Hydroxyalkenal oxPLs adduct with protein lysyl ε-amino groups to produce carboxyalkyl pyrroles after phospholipolysis.
Figure 2. Generation of truncated phospholipids by oxidative fragmentation and chemical synthesis
Oxidative cleavage of PAPC generates POVPC that is further oxidized to PGPC. Mass spectroscopidc comparison with authentic samples (prepared by unambiguous chemical syntheses) of oxPLs generated from PAPC, as well as derivatives generated by reactions of the oxPLs with methoxyamine, sodium borohydride or pentaflourobenzyl bromide, confirmed their molecular structures. Analogous oxidative cleavage of 1-palmityl-2-linoleyl-_sn_-glycero-3-phosphocholine (PLPC) generates PONPC that is further oxidized to PAzPC.
Figure 3
Panel A. POVPC activates aortic smooth muscle cell proliferation: POVPC actives UDP-galactose:glucosylceramide(β1→4)galactosyltransferase (GalT-2) to produce lactosylceramide (LacCer) that provokes the production of superoxide, presumably by stimulating NADPH oxidase (Nox). This leads to activation of the cytosolic transcription factor p44 MAPK. The phosphorylated form of p44 MAPK translocates to the nucleus where it promotes expression of the protooncogene c-fos and proliferating cell nuclear antigen (PCNA) resulting in cell proliferation. Panel B. PGPC Enhances neutrophil-EC interaction, monocyte maturation and, with PONPC and PAzPC, promotes blood coagulation, and POVPC inhibits LPS-induced expression of E-Selectin: PGPC promotes expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin on the apical EC surface, weakly activates PPARγ (2 fold at 6.6 μg/ml) in the nucleus of monocytes, and triggers a bifurcated cascade initiated by a rise in cytosolic calcium concentration resulting in expression of tissue factor (TF) on ECs. POVPC exhibits none of these activities, but rather inhibits LPS-induced E-selectin expression. The procoagulant activity of TF is amplified by the inhibition of tissue factor pathway inhibitor (TFPI) by PONPC and PAzPC, homologues of POVPC and PGPC respectively, neither of which inhibit TFPI. Panel C. POVPC, PGPC & HAzPC initiate apoptosis: OxPC enter cells through TMEM30a. They disrupt the mitochondrial membrane, cause release of cytochrome c and AIF that enter the nucleus and induce expression of caspase resulting in apoptosis.
Figure 4. LC-MS/MS isolation and structural characterization of epoxyisoprostanes
Reconstructed selected ion chromatograms from ESI-LC/MS of oxPAPC showing peaks for 1-palmityl-2-(epoxy-isoprostane)-_sn_-glycero-3-phosphorylcholines (PEIPCs) m/z 828.5 (solid line) and 1-palmityl-2-(epoxycyclopentenone)-_sn_-glycero-3-phosphorylcholines (PECPCs) m/z 810.5 (dashed line) for (panel A) normal phase HPLC separation of oxPAPC and (panel B) reverse phase separation of the fraction shaded in panel A (adapted from J biol chem. 1999;274:24787–24798). Panel C: the structure of one of the products, 5,6-PEIPC, from oxidation of PAPC was established by analysis of its mass and ultraviolet spectra as well as those of the product of dehydration (5,6-PECPC) and phospholipolysis (5,6-EC), and reduction with sodium borodeuteride.
Figure 5. Dehydrative cyclization of isoprostanoid hydroperoxy endoperoxides generates epoxyisoprostanes
Oxidation of PAPC generates four structurally isomeric mixtures of stereoisomeric PEIPCs. Mass spectra of the epoxyisoprostane (EI) free acids or the derived EI-diols allowed identification of structural isomers. Dehydration of PEIPCs delivers the corresponding 1-palmityl-2-(epoxycyclopentenone)-_sn_-glycero-3-phosphorylcholines (PECPCs). The 11,12-PECPC, a cyclopentenone, is 185% more potent than its hydroxy cyclopentanone precursor 11,12-PEIPC for transcriptional activation of PPARα.
Figure 6. Panel A. Alternative oxidative fragmentations generate “mirror image” γ-hydroxyalkenals
Oxidative cleavage of PAPC generates HOOA-PC, a γ-hydroxyalkenal oxPL analogue of HNE. Panel B. Aduction of γ-hydroxyalkenals with primary amines generates carboxyalkylpyrroles: Adduction of γ-hydroxyalkenal phospholipids with primary amino groups, e.g., of proteins, generates ω–carboxyalkyl pyrroles. Panel C. CEP and VEGF Mediated Angiogenesis: ω–Carboxyethylpyrrole (CEP) promotes MyD88-dependant GTP loading (activation) of Rac1 through binding with TLR1/2 leading to NFkB stimulation, integrin expression and proangiogenic activities. The VEGF/VEGFR pathway is an independent parallel integrin-mediated proangiogenic pathway. The tripeptide RGD blocks both pathways.
Figure 7. Chemical synthesis enabled structural and biological characterization of OxPCCD36
HPLC analysis and fractionation of CD36 ligands generated during oxidation of PAPC. OxPAPC was fractionated by preparative reverse phase HPLC and monitored by evaporative light scattering (panel a bottom graph). Three fractions (I-III in panel A upper graph) were found to contain CD36 ligands. Panel B: (top) CD36 binding, LC-MS/MS detection of (middle) HOOA-PC and HOOA-PC methoxime (bottom). Panel C: structures of CD36 binding oxPL shown to be present in fractions isolated from (left) oxPAPC or (right) oxPLPC. Adapted from J biol chem (2002) 38503–16.
Figure 8. Prothrombotic activities of oxidatively-truncated phospholipids
Panel A: Oxidatively truncated phospholipids promote thrombosis by activating platelets through the scavenger receptor CD36 and PAFR, inducing expression of procoagulant tissue factor (TF) on vascular endothelium, and blocking inhibition of that activity by tissue factor pathway inhibitor (TFPI). Panel B: Oxidatively truncated ether phospholipids, referred to collectively as oxPAFPAFR are generated by oxidative cleavage of PUFA esters of lyso-PAF (LPAF) and cause transient (peak in ~100 sec) elevations in Ca+2 levels ranging from 1.5x to more than 3x.
References
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