In vitro study of the cytotoxicity of isolated oxidized lipid low-density lipoproteins fractions in human endothelial cells: relationship with the glutathione status and cell morphology

Toxic effects of oxidized lipid compounds contained in oxidized LDL to endothelial cells are involved in the pathogenesis of atherosclerosis. Glutathione (GSH) plays an important role in the redox status of the cell and in the protective effect against oxidant injuries. However, little is known about the respective effect of these different oxidized lipid compounds toward cytotoxicity and GSH status of the cell. In this report, we isolated by high-performance liquid chromatography oxidized lipid compounds from low-density lipoproteins (LDL) oxidized by copper and we examined their effects on cultured endothelial cells. Cytotoxicity and GSH status were determined after incubation of endothelial cells with crude LDL or isolated lipid fractions derived from cholesterol, phospholipids, or cholesteryl esters. Their effects on cell morphology were also assessed. Oxidized lipids coming from cholesteryl esters (hydroperoxides or short-chain polar derivatives) induced a slight but significant GSH depletion without inducing cytotoxicity. The same species coming from phospholipids induced a more pronounced GSH depletion and a cytotoxic effect which is only present for the more polar compounds (short-chain polar derivatives) and corresponding to a total GSH depletion. In contrast, fractions containing oxysterols had a larger cytotoxic effect than their effect on GSH depletion suggesting that their cytotoxic effects are mediated by a GSH-independent pathway. All together, these data suggest that LDL-associated oxidized lipids present in copper-oxidized LDL exert cytotoxicity by an additional or synergistic effect on GSH depletion, but also by another mechanism independent of the redox status of the cell.     

Oxidized phospholipids as modulators of inflammation in atherosclerosis

PURPOSE OF REVIEW: This review will summarize recent evidence demonstrating that biologically active phospholipid oxidation products modulate inflammatory reactions. RECENT FINDINGS: Structural identification of new biologically active oxidized phospholipids and the finding that they can also be formed at inflammatory sites other than the atherosclerotic lesion have expanded the potential role of these compounds in inflammation beyond atherogenesis. Various signaling pathways are induced by oxidized phospholipids, leading to the expression of inflammatory genes by mechanisms that differ from those mediated by the classic inflammatory agonists tumor necrosis factor or lipopolysaccharide. Furthermore, oxidized phospholipids can bind to pattern recognition molecules and thus potently influence inflammation and immune responses during host defense. SUMMARY: During inflammatory processes biologically active lipid oxidation products accumulate that modulate the inflammatory process and may determine the fate and outcome of the body's reaction in acute inflammation during host defense. Oxidized phospholipids may induce and propagate chronic inflammatory processes; however, evidence is accumulating that cells and tissues respond towards these oxidatively formed stress signals also by activation of anti-inflammatory, cytoprotective reactions.     

Oxidized phospholipids: from molecular properties to disease

Oxidized lipids are generated from (poly)unsaturated diacyl- and alk(en)ylacyl glycerophospholipids under conditions of oxidative stress. The great variety of reaction products is defined by the degree of modification, hydrophobicity, chemical reactivity, physical properties and biological activity. The biological activities of these compounds may depend on both, the recognition of the particular molecular structures by specific receptors and on the unspecific physical and chemical effects on their target systems (membranes, proteins). In this review, we aim at highlighting the molecular features that are essential for the understanding of the biological actions of pure oxidized phospholipids. Firstly, their chemical structures are described as a basis for an understanding of their physical and (bio)chemical properties in membrane- and protein-bound form. Secondly, the biological activities of oxidized phospholipids are discussed in terms of their unspecific effects on the membrane level as well as their potential interactions with specific targets (receptors) affecting a large set of (signaling) molecules. Finally, the role of oxidized phospholipids as important mediators in pathophysiology is discussed with emphasis on atherosclerosis.     

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4: roles for CD14, LPS-binding protein, and MD2 as targets for specificity of inhibition

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysaccharide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-alpha production, IkappaBalpha degradation, p38 MAPK phosphorylation, and NF-kappaB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I.C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from approximately 30 microm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.     

Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase

Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A(2) (cPLA(2)). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor alpha (PPARalpha) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARalpha null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA(2) and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases.     

Inhibition of bacterial lipopolysaccharide-induced inflammatory effects by lipid peroxidation products

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (PAPC) was earlier shown to inhibit inflammatory effects of the bacterial endotoxin lipopolisacharide (LPS). We studied the antiendotoxin activity of other classes of oxidized phospholipids carrying different polar groups and fatty acids. Oxidized phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidic acid inhibited the LPS-induced expression of E-selectin on the surface of human endothelial cells. The type of esterified polyunsaturated fatty acid was not essential for inhibition of the LPS effects. Native unoxidized phospholipids did not influence the effects of LPS. Thus, the anti-endotoxin activity of oxidized phospholipids crucially depends on the presence of an oxidation-modified fatty acid residue.     

Oxidized phospholipids in minimally modified low density lipoprotein induce apoptotic signaling via activation of acid sphingomyelinase in arterial smooth muscle cells

Oxidized phospholipids, including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), typically present in minimally modified low density lipoprotein, have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses (inflammation, proliferation, or cell death). It was the aim of this study to understand their impact on intracellular signal transduction pathways that are responsible for these biological effects. We found that in arterial smooth muscle cells, PGPC and POVPC activated sphingomyelinases, in particular the acid isoform, which is known to participate in the very early phase of apoptotic stress responses. In addition, mitogen-activated protein kinases, which are involved in induction of stress response and apoptosis were phosphorylated (activated). Finally, activation of caspase 3 was observed, showing that stimulation of smooth muscle cells with POVPC and PGPC is associated with apoptosis. Stimulation of all these enzymes by the oxidized phospholipids almost perfectly matched their activation by minimally modified LDL. Consequently, these phospholipids seem to be responsible for the effect of this particle on cell signaling. Survival and proliferation pathways including NF-kappa B or AKT kinase were not induced by POVPC and PGPC. Experiments with a specific inhibitor of acid sphingomyelinase named NB6 showed that this enzyme plays a central role in mediating the apoptotic effects of the oxidized lipids. Thus, we conclude that modified phospholipids induce signaling cascades via activation of acid sphingomyelinase finally leading to apoptosis of smooth muscle cells, which is a detrimental process in the development of atherosclerosis.     

Analysis of oxidized and chlorinated lipids by mass spectrometry and relevance to signalling

Oxidized and chlorinated phospholipids are generated under inflammatory conditions and are increasingly understood to play important roles in diseases involving oxidative stress. MS is a sensitive and informative technique for monitoring phospholipid oxidation that can provide structural information and simultaneously detect a wide variety of oxidation products, including chain-shortened and -chlorinated phospholipids. MSn technologies involve fragmentation of the compounds to yield diagnostic fragment ions and thus assist in identification. Advanced methods such as neutral loss and precursor ion scanning can facilitate the analysis of specific oxidation products in complex biological samples. This is essential for determining the contributions of different phospholipid oxidation products in disease. While many pro-inflammatory signalling effects of oxPLs (oxidized phospholipids) have been reported, it has more recently become clear that they can also have anti-inflammatory effects in conditions such as infection and endotoxaemia. In contrast with free radical-generated oxPLs, the signalling effects of chlorinated lipids are much less well understood, but they appear to demonstrate mainly pro-inflammatory effects. Specific analysis of oxidized and chlorinated lipids and the determination of their molecular effects are crucial to understanding their role in disease pathology.     

Chemistry of phospholipid oxidation

The oxidation of lipids has long been a topic of interest in biological and food sciences, and the fundamental principles of non-enzymatic free radical attack on phospholipids are well established, although questions about detail of the mechanisms remain. The number of end products that are formed following the initiation of phospholipid peroxidation is large, and is continually growing as new structures of oxidized phospholipids are elucidated. Common products are phospholipids with esterified isoprostane-like structures and chain-shortened products containing hydroxy, carbonyl or carboxylic acid groups; the carbonyl-containing compounds are reactive and readily form adducts with proteins and other biomolecules. Phospholipids can also be attacked by reactive nitrogen and chlorine species, further expanding the range of products to nitrated and chlorinated phospholipids. Key to understanding the mechanisms of oxidation is the development of advanced and sensitive technologies that enable structural elucidation. Tandem mass spectrometry has proved invaluable in this respect and is generally the method of choice for structural work. A number of studies have investigated whether individual oxidized phospholipid products occur in vivo, and mass spectrometry techniques have been instrumental in detecting a variety of oxidation products in biological samples such as atherosclerotic plaque material, brain tissue, intestinal tissue and plasma, although relatively few have achieved an absolute quantitative analysis. The levels of oxidized phospholipids in vivo is a critical question, as there is now substantial evidence that many of these compounds are bioactive and could contribute to pathology. The challenges for the future will be to adopt lipidomic approaches to map the profile of oxidized phospholipid formation in different biological conditions, and relate this to their effects in vivo. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.     

Inhibition of LPS- and CpG DNA-induced TNF-alpha response by oxidized phospholipids

Lipid oxidation is commonly seen in the innate immune response, in which reactive oxygen intermediates are generated to kill pathogenic microorganisms. Although oxidation products of phospholipids have generally been regarded to play a role in a number of chronic inflammatory processes, several studies have shown that oxidized phospholipids inhibit the LPS-induced acute proinflammatory response in cultured macrophages and endothelial cells. We report in this study that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), but not nonoxidized PAPC, significantly inhibits the LPS-induced TNF-alpha response in intact mice. Oxidized PAPC also inhibits the 2'-deoxyribo(cytidine-phosphate-guanosine) (CpG) DNA-induced TNF-alpha response in cultured macrophages and intact mice. To elucidate the mechanisms of action, we show that oxidized PAPC, but not nonoxidized PAPC, inhibits the LPS- and CpG-induced activation of p38 MAPK and the NF-kappaB cascade. These results suggest a role for oxidized lipids as a negative regulator in controlling the magnitude of the innate immune response. Further studies on the mechanisms of action may lead to development of a new type of anti-inflammatory drug for treatment of acute inflammatory diseases such as sepsis.     

Oxidized phospholipids as biomarkers of tissue and cell damage with a focus on cardiolipin

Oxidized phospholipid species are important, biologically relevant, lipid signaling molecules that usually exist in low abundance in biological tissues. Along with their inherent stability issues, these oxidized lipids present themselves as a challenge in their detection and identification. Often times, oxidized lipid species can co-chromatograph with non-oxidized species making the detection of the former extremely difficult, even with the use of mass spectrometry. In this study, a normal-phase and reverse-phase two dimensional high performance liquid chromatography (HPLC)-mass spectrometric system was applied to separate oxidized phospholipids from their non-oxidized counterparts, allowing unambiguous detection in a total lipid extract. We have utilized bovine heart cardiolipin as well as commercially available tetralinoleoyl cardiolipin oxidized with cytochrome c (cyt c) and hydrogen peroxide as well as with lipoxygenase to test the separation power of the system. Our findings indicate that oxidized species of not only cardiolipin, but other phospholipid species, can be effectively separated from their non-oxidized counterparts in this two dimensional system. We utilized three types of biological tissues and oxidative insults, namely rotenone treatment of lymphocytes to induce mitochondrial damage and cell death, pulmonary inhalation exposure to single walled carbon nanotubes, as well as total body irradiation, in order to identify cardiolipin oxidation products, critical to the cell damage/cell death pathways in these tissues following cellular stress/injury. Our results indicate that selective cardiolipin (CL) oxidation is a result of a non-random free radical process. In addition, we assessed the ability of the system to identify CL oxidation products in the brain, a tissue known for its extreme complexity and diversity of CL species. The ability of the two dimensional HPLC-mass spectrometric system to detect and characterize oxidized lipid products will allow new studies to be formulated to probe the answers to biologically important questions with regard to oxidative lipidomics and cellular insult. This article is part of a Special Issue entitled: Oxidized phospholipids - their properties and interactions with proteins.     

Oxidized phospholipids: Biomarker for cardiovascular diseases

Biologically active oxidized phospholipids can initiate and modulate many of the cellular events attributed to inflammation leading to atherosclerosis. Produced by enzymatic or non-enzymatic processes, these molecules interact with various cells via specific receptors and in general give rise to inflammatory signals. There is considerable evidence that oxidized phospholipids accumulate in vivo and play significant roles in atherosclerosis and thrombosis, suggesting that oxidized phospholipids could be biomarkers that reflect the global extent of these diseases in vivo. Thus, understanding the biosynthetic pathways, receptor specificity and signaling processes of oxidized phospholipids is important in understanding atherosclerosis, thrombosis and related inflammatory diseases.     

The oxidized phospholipids POVPC and PGPC inhibit growth and induce apoptosis in vascular smooth muscle cells

Oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are typically present in oxidatively modified low density lipoprotein (oxLDL) and have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses like inflammation, proliferation, or cell death. The aim of this study was to elicit the type and outcome of the cellular response of vascular smooth muscle cells (VSMC) upon treatment with POVPC and PGPC. Both oxidized phospholipids led to inhibition of cell proliferation and showed cytotoxic effects in VSMC. Several morphological criteria, the presence of typical DNA fragments, and a phosphatidylserine shift towards the outer leaflet of the cell membrane revealed that apoptosis was the predominant mode of cell death. In all experiments, POVPC was found to be a more potent inducer of apoptosis than PGPC. Interestingly, in the presence of high levels of serum in the growth media the proapoptotic but not the antiproliferative effects of both oxidized phospholipids were abolished. Thus, we conclude that under low serum conditions both intact POVPC and PGPC are proapoptotic mediators. Under high serum conditions, these lipids are hydrolyzed and the resultant lipid mixture containing the degradation products is no longer apoptotic but antiproliferative. Thus, the direct and indirect effects of POVPC and PGPC on cell viability may account for the detrimental actions of oxLDL on VSMC.     

Oxidized low density lipoprotein and innate immune receptors

PURPOSE OF REVIEW: Atherosclerosis is now recognized as a chronic inflammatory disease. This review discusses recent literature reporting that innate immune receptors bind oxidatively modified LDL and its many oxidized moieties and consequently modulate the atherogenic process. These innate pattern recognition receptors are known to play a central role in pro-inflammatory responses to bacteria by binding pathogen-associated molecular patterns. It is hypothesized that oxidized LDL exposes similar molecular patterns recognized by receptors of innate immunity. RECENT FINDINGS: Minimally modified LDL and its oxidized phospholipids have been found to bind to CD14 or activate Toll-like receptors on macrophages. In turn, various biological activities have been induced, including the stimulation of cytoskeletal rearrangements that alter phagocytic activity and the stimulation of cytokine secretion, such as IL-8. These findings link modified LDL with innate pattern recognition receptors, such as those involved in the lipopolysaccharide signaling pathway. Human epidemiological studies support the involvement of CD14 and TLR4 in cardiovascular diseases. Oxidized LDL has also been demonstrated to bind to C-reactive protein, an opsonic molecule activating classic complement pathway and Fcgamma receptor endocytosis. These data suggest that C-reactive protein may not only be a strong predictor of clinical disease, but may also play a role in atherogenesis. Recent data on other innate immune receptors are discussed in the context of their potential interactions with oxidized LDL and atherogenesis. SUMMARY: Recent findings suggest that oxidized forms of LDL interact with innate immune receptors. Further studies are needed to identify the role of these interactions in inflammation and atherosclerosis.     

High-density lipoproteins protect endothelial cells from apoptosis induced by oxidized low-density lipoproteins

Summary Endothelial lesion by oxidized low-density liproproteins (LDL) is one of the first stages in the development of atherosclerosis. The effect of these lipoproteins can range from a functional lesion of the endothelium to death of the endothelial cells by apoptosis. High-density lipoproteins (HDL) are one of the factors which can have a protective effect against the development of atheromatous plaques. The aim of this study is to establish whether the death of endothelial cells by apoptosis induced by oxidized LDLs is prevented by HDLs. ECV304 endothelial cells and bovine aorta endothelial cells were incubated with native LDLs, oxidized LDLs, and a combination of both oxidized LDLs and HDLs. Oxidized LDLs caused a significant increase of mortality mainly by apoptosis. However, when HDLs were added together with oxidized LDLs the percentage of total mortality, the degree of lipoprotein oxidation in the medium, and the percentage of cells in apoptosis were all significantly decreased. HDLs protect against the cytotoxicity of oxidized LDLs possibly by preventing the propagation of the oxidative chain in these lipoproteins.Abbreviations LDL low-density lipoproteins - HDL high-density lipoproteins - BAEC bovine aortic endothelial cell - TBARS thiobarbituric acid-reactive substances     

The functional effects of physical interactions among Toll-like receptors 7, 8, and 9

Toll-like receptors (TLRs) TLR1, TLR2, TLR4, and TLR6 are evolutionarily conserved, highly homologous, and localized to plasma membranes of host cells and recognize pathogen-associated molecular patterns (PAMPs) derived from bacterial membranes. These receptors cooperate in a pairwise combination to elicit or inhibit the inflammatory signals in response to certain PAMPs. The other TLRs that are evolutionarily closely related and highly homologous are TLR7, TLR8, and TLR9. They are all confined to the membranes of endosomes and recognize similar molecular structures, the oligonucleotide-based PAMPs. However, the cooperative interactions among these receptors that may modulate the inflammatory signaling in response to their cognate agonists are not reported. We report here for the first time the functional effects of one TLR on the other among TLR7, TLR8, and TLR9. The results indicate that TLR8 inhibits TLR7 and TLR9, and TLR9 inhibits TLR7 but not vice versa in HEK293 cells transfected with TLRs in a pairwise combination. This is concluded by selectively activating one TLR over the other by using small molecule TLR agonists. We also show that these inhibitory interactions are the result of direct or indirect physical interactions between the TLRs. The murine TLR8 that does not respond to any known human TLR8 agonists also inhibits both murine and human TLR7. The implications of the inhibitory interactions among these TLRs in host-pathogen recognition and subsequent inflammatory responses are not obvious. However, given the complexity in expression pattern in a particular cell type and the variation in distribution and response to different pathogens and stress signals in different cell types, the inhibitory physical interactions among these TLRs may play a role in balancing the inflammatory outcome from a given cell type to a specific challenge.