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Oxidized phospholipids (OxPLs) are known to induce in cultured cells multiple biological effects that are potentially relevant to pathology. However, until recently it was not clear if accumulation of OxPLs plays a causative role in disease or is an epiphenomenon. Recent progress in identification of molecular targets of OxPLs led to development of new in vivo models that clearly demonstrated the importance of these lipid mediators in pathology. This review discusses currently known receptor and non–receptor-dependent mechanisms triggering biological action of OxPLs. Rapidly accumulating evidence suggests that some of these mechanisms represent promising targets for therapy.

Circulating oxidized phospholipids are increasingly recognized as biomarkers of atherosclerosis. Clinical association studies have been mainly performed using an immune assay based on monoclonal antibody E06, which recognizes a variety of molecular species of oxidized phosphatidylcholine (OxPC) in lipoproteins, cell membranes or covalently bound to plasma proteins. Accumulating evidence shows that individual molecular species of OxPC demonstrate different biological activities and have different half-life times. Therefore, it is likely that certain molecular species can be associated with pathology more strongly than others. This hypothesis can only be tested using LC-MS/MS allowing quantification of individual molecular species of OxPCs. In order to ensure that laborious LC-MS/MS methods do not simply replicate the results of a technically simpler E06-OxPCs assay, we have performed relative quantification of 8 truncated molecular species of OxPCs in plasma of 132 probands and compared the data with the results of the E06-OxPCs and OxLDL assays. We have found a strong correlation between individual molecular species of OxPCs but only a weak correlation of LC-MS/MS-OxPCs data with the E06-OxPCs assay and no correlation with the OxLDL assay. Furthermore, in contrast to the results of E06-OxPCs or OxLDL assays, 7 out of 8 OxPC species were associated with hypertension. The data suggest that the results of the LC-MS/MS-OxPCs assay do not replicate the results of two ELISA-based lipid oxidation tests and therefore may produce additional diagnostic information. These findings necessitate development of simplified mass spectrometric procedures for high-throughput and affordable analysis of selected molecular species of OxPCs.

International audience; Oxidized phospholipids (OxPLs) containing enzymatically or non-enzymatically oxidized fatty acids (oxylipins) are increasingly recognized as lipid mediators involved in pathogenesis of diseases. Further understanding of structure-activity relationship and molecular mechanisms activated by OxPLs is hampered by the complexity of synthesis of individual molecular species. Although dozens of individual free oxylipins are commercially available, their attachment to the phospholipid scaffold requires relatively harsh conditions during activation of carboxy-group, which may lead to decomposition of unstable oxylipins. Furthermore, additional protection-deprotection steps are required for oxylipins containing hydroxy-groups. In this work we describe synthesis of OxPLs containing oxylipins bound at the sn-2-position via an amide-bond that is characteristic of sphingophospholipids. Activation of oxylipins and attachment to the phospholipid scaffold are performed under mild conditions and characterized by high yield. Hydroxy-groups of oxylipins do not interfere with reactions and therefore no protection/deprotection steps are needed. In order to prevent oxylipin migration, a fatty acid residue at the sn-1 was bound through an alkyl bond, which is a common bond present in a large proportion of naturally occurring phospholipids. An additional advantage of combining alkyl and amide bonds in a single phospholipid molecule is that both types of bonds are phospholipase A1/A2-resistant, which may be expected to improve biological stability of OxPLs and thus simplify analysis of their effects. As proof of principle, several alkyl-amide oxidized phosphatidylcholines (OxPCs) containing either linear or prostane ring oxylipins have been synthesized. Importantly, we show here that alkyl-amide-OxPCs demonstrated biological activities similar to those of di-acyl-OxPCs. Alkyl-amide-OxPCs inhibited pro-inflammatory action of LPS and increased endothelial cellular barrier in vitro and in mouse models. The effects of alkyl-amide and di-acyl-OxPCs developed in a similar range of concentrations. We hypothesize that alkyl-amide-OxPLs may become a useful tool for deeper analysis of the structure-activity relationship of OxPLs.

Processed and ready-to-eat foods become routinely consumed resulting in a sharp rise of sugar intake in people's daily diets. The inclusion of fresh fruits and vegetables rich in (poly)phenols has been encouraged by the World Health Organization (WHO) as part of the daily choices to ameliorate endothelial dysfunction and ease the socio-economic burden of diabetes. Research in Food, Nutrition, and Cell Metabolism areas is revealing that the health benefits of (poly)phenol-rich foods go beyond their antioxidant properties and are in fact key modulators of redox and glycaemia status, and inflammatory response contributing to improved endothelial function and vascular health in diabetes. Other beneficial aspects include appetite modulation, regulation of hydrolytic enzymes involved in sugar and lipid metabolism, and mediation of cell-cell aggregation events. This work overviews the current knowledge on the biological properties of ingested (poly)phenols in cultured endothelial cells with emphasis on the circulating (poly)phenols, providing support to (poly)phenol-rich diets as alternatives to drug-based therapies in the prevention, treatment, and management of diabetes. A critical evaluation on the caveats and challenges involve in current experimental cell-based designs and approaches adopted is also discussed.

International audience; A comprehensive and standardized system to report lipid structures analyzed by MS is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e., annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labeled compounds in metabolic labeling experiments or as internal standards. This update on lipid classification, nomenclature, and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.

We have reported recently that inactivation of the essential autophagy-related gene 7 (Atg7) in keratinocytes has little or no impact on morphology and function of the epidermal barrier in experimental animals. When these mice aged, mutant males, (Atg7 ΔKC), developed an oily coat. As the keratin 14 promoter driven cre/LoxP system inactivates floxed Atg7 in all keratin 14 (K14) expressing cells, including sebocytes, we investigated whether the oily hair phenotype was the consequence of changes in function of the skin sebaceous glands. Using an antibody to the GFP-LC3 fusion protein, autophagosomes were detected at the border of sebocyte disintegration in control but not in mutant animals, suggesting that autophagy was (a) active in normal sebaceous glands and (b) was inactivated in the mutant mice. Detailed analysis established that dorsal sebaceous glands were about twice as large in all Atg7 ΔKC mice compared to those of controls (Atg7 F/F), and their rate of sebocyte proliferation was increased. In addition, male mutant mice yielded twice as much lipid per unit hair as age-matched controls. Analysis of sebum lipids by thin layer chromatography revealed a 40% reduction in the proportion of free fatty acids (FFA) and cholesterol, and a 5-fold increase in the proportion of fatty acid methyl esters (FAME). In addition, the most common diester wax species (58-60 carbon atoms) were increased, while shorter species (54-55 carbon atoms) were under-represented in mutant sebum. Our data show that autophagy contributes to sebaceous gland function and to the control of sebum composition.

Receptor tyrosine kinase c-Kit and its ligand stem cell factor (SCF) regulate resident vascular wall cells and recruit circulating progenitors. We tested whether SCF may be induced by oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) known to accumulate in atherosclerotic vessels. Gene expression analysis demonstrated OxPAPC-induced upregulation of SCF mRNA and protein in different types of endothelial cells (ECs). Elevated levels of SCF mRNA were observed in aortas of ApoE−/− knockout mice. ECs produced biologically active SCF because conditioned medium from OxPAPC-treated cells stimulated activation (phosphorylation) of c-Kit in naïve ECs. Induction of SCF by OxPAPC was inhibited by knocking down transcription factor NRF2. Inhibition or stimulation of NRF2 by pharmacological or molecular tools induced corresponding changes in SCF expression. Finally, we observed decreased levels of SCF mRNA in aortas of NRF2 knockout mice. We characterize OxPLs as a novel pathology-associated stimulus inducing expression of SCF in endothelial cells. Furthermore, our data point to transcription factor NRF2 as a major mediator of OxPL-induced upregulation of SCF. This mechanism may represent one of the facets of pleiotropic action of NRF2 in vascular wall. Electronic supplementary material The online version of this article (10.1007/s10456-017-9590-5) contains supplementary material, which is available to authorized users.

Unlike other agonists that cause transient endothelial cell (EC) response, the products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) oxidation that contain cyclopenthenone groups, which recapitulate prostaglandin-like structure, cause sustained enhancement of the pulmonary EC barrier. The mechanisms that drive the sustained effects by oxidized PAPC (OxPAPC) remain unexplored. On the basis of the structural similarity of isoprostanoid moieties that are present in full-length oxygenated PAPC species, we used an inhibitory approach to perform the screening of prostanoid receptors as potential candidates that mediate OxPAPC effects. Results show that only prostaglandin E receptor-4 (EP4) was involved and mediated the sustained phase of the barrier-enhancing effects of OxPAPC that are associated with the activation of Rac GTPase and its cytoskeletal targets. EC incubation with OxPAPC also induced EP4 mRNA expression in pulmonary ECs and lung tissue. EP4 knockdown using gene-specific small interfering RNA did not affect the rapid phase of OxPAPC-induced EC barrier enhancement or the protective effects against thrombin-induced EC permeability, but abolished the advanced barrier enhancement phase and suppressed the protective effects of OxPAPC against more sustained EC barrier dysfunction and cell inflammatory response caused by TNF-α. Endothelial-specific knockout of the EP4 receptor in mice attenuated the protective effect of intravenous OxPAPC administration in the model of acute lung injury caused by intratracheal injection of LPS. Taken together, these results demonstrate a novel role for prostaglandin receptor EP4 in the mediation of barrier-enhancing and anti-inflammatory effects caused by oxidized phospholipids.—Oskolkova, O., Gawlak, G., Tian, Y., Ke, Y., Sarich, N., Son, S., Andreasson, K., Bochkov, V. N., Birukova, A. A., Birukov, K. G. Prostaglandin E receptor-4 receptor mediates endothelial barrier–enhancing and anti-inflammatory effects of oxidized phospholipids.

Rationale: Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) generates a group of bioactive oxidized phospholipid products with a broad range of biological activities. Barrier-enhancing and anti-inflammatory effects of OxPAPC on pulmonary endothelial cells are critical for prevention of acute lung injury caused by bacterial pathogens or excessive mechanical ventilation. Anti-inflammatory properties of OxPAPC are associated with its antagonistic effects on Toll-like receptors and suppression of RhoA GTPase signaling. Objective: Because OxPAPC exhibits long-lasting anti-inflammatory and lung-protective effects even after single administration in vivo, we tested the hypothesis that these effects may be mediated by additional mechanisms, such as OxPAPC-dependent production of anti-inflammatory and proresolving lipid mediator, lipoxin A4 (LXA4). Methods and Results: Mass spectrometry and ELISA assays detected significant accumulation of LXA4 in the lungs of OxPAPC-treated mice and in conditioned medium of OxPAPC-exposed pulmonary endothelial cells. Administration of LXA4 reproduced anti-inflammatory effect of OxPAPC against tumor necrosis factor-α in vitro and in the animal model of lipopolysaccharide-induced lung injury. The potent barrier-protective and anti-inflammatory effects of OxPAPC against tumor necrosis factor-α and lipopolysaccharide challenge were suppressed in human pulmonary endothelial cells with small interfering RNA–induced knockdown of LXA4 formyl peptide receptor-2 (FPR2/ALX) and in mFPR2 −/− (mouse formyl peptide receptor 2) mice lacking the mouse homolog of human FPR2/ALX. Conclusions: This is the first demonstration that inflammation- and injury-associated phospholipid oxidation triggers production of anti-inflammatory and proresolution molecules, such as LXA4. This lipid mediator switch represents a novel mechanism of OxPAPC-assisted recovery of inflamed lung endothelium.

Products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine oxidation bearing cyclopentenone modifications of arachidonoyl moiety exhibit pronounced protective effects on pulmonary endothelial cell (EC) barrier. On the other hand, prostacyclin and its FDA-approved stable synthetic analog, iloprost, also protect lung vascular EC and attenuate lung inflammation caused by bacterial wall lipopolysaccharide (LPS). In this study, we generated phospholipase resistant synthetic phospholipid with arachidonic acid moiety replaced by iloprost (ILO-PC) and investigated its biological effects. In comparison to free ILO, ILO-PC caused sustained EC barrier enhancement. Both, ILO and ILO-PC effects were mediated by activation of Rap1 and Rac1 GTPases and their cytoskeletal effectors PAK1 and cortactin and enhancement of VE-cadherin adherens junctions. ILO and ILO-PC equally efficiently suppressed acute, Rho GTPase-dependent EC hyper-permeability caused by thrombin. However, ILO-PC exhibited more sustained barrier-protective and anti-inflammatory effects in the model of chronic EC dysfunction caused by LPS. ILO-PC also was more potent inhibitor of NFκB signaling and lung vascular leak in the murine model of LPS-induced acute lung injury (ALI). Optical imaging of lungs of LPS-challenged animals monitored over 3 days showed more efficient ALI recovery in animals treated with ILO-PC. In conclusion, this study describes a novel synthetic phospholipid with barrier-enhancing and anti-inflammatory properties superior to existing prostacyclin analogs, which may be used as a prototype for future development of more efficient treatment for ALI and other vascular leak syndromes.

Correction of barrier dysfunction and inflammation in acute lung injury (ALI) represents an important problem. Previous studies demonstrate barrier-protective and anti-inflammatory effects of bioactive lipid prostacyclin and its stable analog iloprost (ILO). We generated a phospholipase resistant synthetic phospholipid with iloprost attached at the sn-2 position (ILO-PC) and investigated its biological effects. In comparison to free ILO, ILO-PC caused sustained endothelial cell (EC) barrier enhancement, linked to more prolonged activation of Rap1 and Rac1 GTPases and their cytoskeletal and cell junction effectors: cortactin, PAK1, p120-catenin and VE-cadherin. ILO and ILO-PC equally efficiently suppressed acute, Rho GTPase-dependent EC hyper-permeability caused by thrombin. However, ILO-PC exhibited more sustained barrier-protective and anti-inflammatory effects in the model of chronic EC dysfunction caused by bacterial wall lipopolysacharide (LPS). ILO-PC was also more potent inhibitor of NFκB signaling and lung vascular leak in the murine model of LPS-induced ALI. Treatment with ILO-PC showed more efficient ALI recovery over 3 days after LPS challenge than free ILO. In conclusion, this study describes a novel synthetic phospholipid with barrier-enhancing and anti-inflammatory properties superior to existing prostacyclin analogs, which may be used as a prototype for future development of more efficient treatment for ALI and other vascular leak syndromes.

Objective Oxidized phospholipids (OxPLs), which are highly abundant in atherosclerotic lesions, are known to induce electrophilic stress response (ESR). ESR induces cytoprotective genes via the NF-E2-related factor 2 (NRF2) transcription factor. In order to get further insight into the mechanisms of ESR, we studied the role of microRNA (miR)-320a in induction of NRF2-dependent genes by OxPLs. Methods Microarray profiling and qRT-PCR methods were used for measurements of mRNA and miRNA levels. miR-320a levels were changed by transfection with synthetic oligonucleotides. Protein analysis was performed by Western blotting. The functional activity of NRF2 was measured by DNA-binding ELISA. Results Oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) induced miR-320a in endothelial cells. Induction of HO-1, OKL38 and GCLM mRNAs by OxPAPC and sulforaphane was attenuated upon knockdown of miR-320a. In contrast, transfection of ECs with miR-320a mimic oligonucleotide potentiated the effects of OxPAPC and sulforaphane on induction of HO-1, OKL38 and GCLM mRNAs. OxPAPC-induced p38 activation, levels of NRF2 protein and its ability to bind to consensus NRF2 DNA binding site were elevated in ECs transfected with miR-320a mimic. miR-320a positively regulated induction of VEGF mRNA by OxPAPC. Levels of miR-320a and HO-1 and OKL38 mRNAs were elevated in aortas of ApoE knockout mice fed with high fat diet. Manipulation of miR-320a level in ECs did not affect ability of OxPAPC to induce IL-8, COX-2 and MCP-1. Conclusion miR-320a plays important role in induction of expression of HO-1, GCLM and OKL38 upon ESR induced either by OxPAPC or sulforaphane. These observations propose a general role of miR-320a in control of ESR induced by different electrophilic agents.

This is the first report of heat shock protein serving as a cell surface receptor of oxidized phospholipids. It also investigates downstream molecular mechanisms and shows that GRP78 is involved in assembly of S1PR1-Akt-Rac1 signalosome, which is critical for vasoprotective effects of oxidized phospholipids., Vascular integrity and the maintenance of blood vessel continuity are fundamental features of the circulatory system maintained through endothelial cell–cell junctions. Defects in the endothelial barrier become an initiating factor in several pathologies, including ischemia/reperfusion, tumor angiogenesis, pulmonary edema, sepsis, and acute lung injury. Better understanding of mechanisms stimulating endothelial barrier enhancement may provide novel therapeutic strategies. We previously reported that oxidized phospholipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [OxPAPC]) promote endothelial cell (EC) barrier enhancement both in vitro and in vivo. This study examines the initiating mechanistic events triggered by OxPAPC to increase vascular integrity. Our data demonstrate that OxPAPC directly binds the cell membrane–localized chaperone protein, GRP78, associated with its cofactor, HTJ-1. OxPAPC binding to plasma membrane–localized GRP78 leads to GRP78 trafficking to caveolin-enriched microdomains (CEMs) on the cell surface and consequent activation of sphingosine 1-phosphate receptor 1, Src and Fyn tyrosine kinases, and Rac1 GTPase, processes essential for cytoskeletal reorganization and EC barrier enhancement. Using animal models of acute lung injury with vascular hyperpermeability, we observed that HTJ-1 knockdown blocked OxPAPC protection from interleukin-6 and ventilator-induced lung injury. Our data indicate for the first time an essential role of GRP78 and HTJ-1 in OxPAPC-mediated CEM dynamics and enhancement of vascular integrity.

A diverse group of bioactive lipids are generated from the oxidation of phospholipids during various pathological conditions such as lung injury, sepsis, and autoimmune diseases. An elevated level of circulating oxidized phospholipids with potent inflammatory activities has been recognized as a hallmark of various diseases including atherosclerosis. On the other hand, the oxidation of a principal membrane phospholipid 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine (PAPC) results in the formation of a heterogeneous mixture of full length and fragmented oxidized species with both beneficial and detrimental effects on pulmonary endothelium. Among these, the extensive studies from this research group have established that full length oxidized species present in oxidized PAPC (OxPAPC) have pronounced endothelial barrier protective and anti‐inflammatory properties. This has been documented in various cellular and animal models of lung injury and inflammation using a wide range of chemical and mechanical stimuli. Moreover, OxPAPC stimulation of endothelial cells generates anti‐inflammatory molecules such as lipoxin that mediate the recovery of inflamed lungs. This review briefly summarizes the current knowledge on OxPAPC‐mediated positive regulation of endothelial barrier function and its potential as a promising therapeutic target for lung injury and inflammation. Practical Applications: This review briefly summarizes the current knowledge on OxPAPC‐mediated positive regulation of endothelial barrier function and its potential as a promising therapeutic target for lung injury and inflammation. Products of phospholipid oxidation may exhibit disruptive or protective effects on vascular endothelium depending on the extent of oxidation. Full length oxidized phospholipids stimulated endothelial barrier recovery and resolution of acute lung injury or sepsis by triggering multiple mechanisms enhancing endothelial barrier properties and diminishing inflammation.

Prostaglandins (PG), the products of cyclooxygenase-mediated conversion of arachidonic acid, become upregulated in many situations including allergic response, inflammation, and injury, and exhibit a variety of biological activities. Previous studies described barrier-enhancing and anti-inflammatory effects of PGE2 and PGI2 on vascular endothelial cells (EC). Yet, the effects of other PG members on EC barrier and inflammatory activation have not been systematically analyzed. This study compared effects of PGE2, PGI2, PGF2α, PGA2, PGJ2, and PGD2 on human pulmonary EC. EC permeability was assessed by measurements of transendothelial electrical resistance and cell monolayer permeability for FITC-labeled tracer. Anti-inflammatory effects of PGs were evaluated by analysis of expression of adhesion molecule ICAM1 and secretion of soluble ICAM1 and cytokines by EC. PGE2, PGI2, and PGA2 exhibited the most potent barrier-enhancing effects and most efficient attenuation of thrombin-induced EC permeability and contractile response, whereas PGI2 effectively suppressed thrombin-induced permeability but was less efficient in the attenuation of prolonged EC hyperpermeability caused by interleukin-6 or bacterial wall lipopolysaccharide, LPS. PGD2 showed a modest protective effect on the EC inflammatory response, whereas PGF2α and PGJ2 were without effect on agonist-induced EC barrier dysfunction. In vivo, PGE2, PGI2, and PGA2 attenuated LPS-induced lung inflammation, whereas PGF2α and PGJ2 were without effect. Interestingly, PGD2 exhibited a protective effect in the in vivo model of LPS-induced lung injury. This study provides a comprehensive analysis of barrier-protective and anti-inflammatory effects of different prostaglandins on lung EC in vitro and in vivo and identifies PGE2, PGI2, and PGA2 as prostaglandins with the most potent protective properties.

Aim: Oxidized phospholipids (OxPL) are the major pathogenic component of oxidized low-density lipoproteins (OxLDL). Endogenous anti-OxPL activity, defined as the ability to neutralize adverse effects of oxidized lipids, may have biomarker potential. Methods & results: Using two anti-OxPL monoclonal antibodies (commercial mAB-E06 and custom mAB-509) we developed a novel ELISA that measures the global capacity of plasma to inactivate OxPL. Preincubation of OxLDL with plasma inhibits its binding of anti-OxPL mABs. This phenomenon (‘masking’) reflects anti-OxPL plasma activity. A pilot clinical application of the assay revealed reduced anti-OxPL activity in hypertension, coronary artery disease, acute coronary syndrome and diabetes. Conclusion: Inadequate anti-OxPL protection may contribute to cardiovascular disease and have biomarker potential in conditions associated with abnormal lipid peroxidation.

Oxidized phospholipids (OxPLs) are increasingly recognized as pleiotropic lipid mediators demonstrating a variety of biological activities. In particular, OxPLs induce electrophilic stress response and stimulate expression of NF-E2-related factor 2 (NRF2)-dependent genes. The mechanisms of NRF2 upregulation in response to OxPLs, however, are incompletely understood. Here we show that upregulation of NRF2 by OxPLs depends on the activity of the CK2 protein kinase. Inactivation of CK2 by chemical inhibitors or gene silencing resulted in diminished accumulation of NRF2 and its target genes, GCLM, HMOX1, and NQO1, downstream in response to OxPLs. Furthermore, inhibition of CK2 suppressed NRF2-dependent induction of ATF4 and its downstream gene VEGF. Thus, inactivation of CK2 in OxPL-treated endothelial cells results in inhibition of the NRF2-ATF4-VEGF axis and is likely to produce antiangiogenic effects. This work characterizes novel cross-talk between CK2 and cellular stress pathways, which may provide additional insights into the mechanisms of beneficial action and side-effects of CK2 inhibitors.

Objective— The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response. Methods and Results— Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation. Conclusion— Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.

Glycerophospholipids represent a common class of lipids critically important for integrity of cellular membranes. Oxidation of esterified unsaturated fatty acids dramatically changes biological activities of phospholipids. Apart from impairment of their structural function, oxidation makes oxidized phospholipids (OxPLs) markers of “modified-self” type that are recognized by soluble and cell-associated receptors of innate immunity, including scavenger receptors, natural (germ line-encoded) antibodies, and C-reactive protein, thus directing removal of senescent and apoptotic cells or oxidized lipoproteins. In addition, OxPLs acquire novel biological activities not characteristic of their unoxidized precursors, including the ability to regulate innate and adaptive immune responses. Effects of OxPLs described in vitro and in vivo suggest their potential relevance in different pathologies, including atherosclerosis, acute inflammation, lung injury, and many other conditions. This review summarizes current knowledge on the mechanisms of formation, structures, and biological activities of OxPLs. Furthermore, potential applications of OxPLs as disease biomarkers, as well as experimental therapies targeting OxPLs, are described, providing a broad overview of an emerging class of lipid mediators. Antioxid. Redox Signal. 12, 1009–1059.

Objective—Oxidized phospholipids (OxPLs) that are abundant in atherosclerotic lesions are increasingly recognized as context-dependent lipid mediators demonstrating both pro- and antiinflammatory activities. Molecular mechanisms of their effects are largely unknown. Here we present novel information on the mechanisms whereby OxPLs modulate activation of TLR4 by lipopolysaccharide (LPS).Methods and Results—We show, using several cell types and various inflammatory genes as readouts, that different classes and molecular species of OxPLs do not stimulate TLR4 but exert prominent inhibitory effects on LPS-induced reactions. Our data demonstrate that binding of OxPLs to the LPS-binding protein (LBP) and CD14 prevents recognition of LPS by these proteins, thus impairing activation of TLR4. In addition, OxPLs inhibited LBP- and CD14-independent activation of TLR4 by the synthetic TLR4 agonist E6020 indicating that in parallel with LBP and CD14, OxPLs target cell-associated steps in TLR4 cascade.Conclusions—Our data suggest that OxPLs inhibit action of LPS via a multi-hit mechanism. These results support the notion that OxPLs are endogenous inhibitors of TLR4 produced in response to oxidative stress.

Lipids are key regulators of immune responses. In this study we investigated the direct impact of oxidized phospholipids (ox-PL) on T cell activation and function. We could demonstrate that ox-PL strongly inhibit proliferation of purified human T cells induced with anti-CD3/CD28 or anti-CD3/CD63 mAb, whereas proliferation of naive T cells from human cord blood was not affected by ox-PL. Unoxidized phospholipids showed no such effect. Inhibition of T cell proliferation by ox-PL was not due to cell death. Moreover, T cell proliferation triggered by PMA/ionomycin activation was not diminished by ox-PL. T cells activated in the presence of ox-PL produced and released low amounts of IFN-gamma and IL-2, whereas IL-4 was only slightly diminished. Ox-PL prevented the expression of de novo synthesized activation markers (CD25, MHC class II) but not expression of CD63 or CD69. We further observed that T cells stimulated in the presence of ox-PL are poorly cytotoxic T cells. Most importantly, T cells activated in the presence of ox-PL failed to proliferate in response to restimulation. This hypo-proliferative state was accompanied with an up-regulation of early growth response gene 3 and Casitas B-lineage lymphoma protein B. Taken together, our results demonstrate that ox-PL are potent and specific regulators of T cell activation and function.

Microparticles (MPs) are released from cells during processes such as apoptosis or during cell activation. These MPs contain phospholipids, proteins and even nucleic acids derived from their parent cells. They are found circulating in plasma but also in tissues such as atherosclerotic plaques. It is thought that MPs contain and transfer tissue factor and can thereby induce blood clotting. In this study we analyzed clot promoting properties of MPs generated from vascular cells in vitro. MPs were generated from endothelial cells (EC), smooth muscles cells (SMC), monocytes (U937), erythrocytes (RBC) or platelets (Pl) by inducing apoptosis or by calcium ionophore activation; they were subsequently isolated by differential centrifugation. Thrombogenicity of the MPs was evaluated using a thrombin generation assay (Technothrombin® TGA) and MP free plasma as substrate. MPs displayed a different thrombin generating potential depending on the parent cells. MPs derived from RBCs (~400nM peak thrombin/105 MPs/ml plasma), ECs (~300nM), SMCs (~300nM) and Pls (~300nM) were more thrombogenic than MPs derived from U937 (~200 nM). In addition EC, SMC and U937 MPs all expressed tissue factor but EC MPs induced thrombin generation in a tissue factor and FVII independent manner. EC MPs even expressed active tissue factor pathway inhibitor and functionally inhibited tissue factor dependent thrombin generation. Since the higher thrombin generation induced by MPs derived from EC as compared U937 derived MPs could not be explained by a different activity of tissue factor, we were interested whether lipids contained in the microparticles could account for the differences in thrombin generation. We therefore analyzed thrombin generation induced by lipids isolated from MPs and parent cells and could show that lipids from EC MPs and SMC MPs exhibited higher thrombin generation than those from U937 MPs. Upon analysis of lipids by thin layer chromatography and mass spectrometry we found that in general microparticles are enriched in cholesterol, sphingomyeline and phosphatidylserine over the parent cells and that EC and SMC MPs were enriched in negatively charged phospholipids (different species of phosphatidylserine and phosphatiylglycerol) as compared to MPs derived from U937 cells. When thrombogenicity was, however, evaluated in vivo by injecting MPs into mice it was found that the highest capability to induce thrombin-antithrombin (TAT) complexes had MPs derived from SMCs; also U937 MPs induced an increase in TAT levels, while EC MPs – although more thrombogenic than U937 MPs in vitro – did not induce TAT complex formation by themselves but were only synergistic in vivo. From these data we conclude that thrombin formation in vivo depends on the initiation of the tissue factor FVII pathway, while the extent of thrombin formation is dependent on negatively charged phospholipids contained to a higher extent in MPs derived e.g. from ECs.

Heme oxygenase-1 (HO-1) is a key enzyme in the cellular response to tissue injury and oxidative stress. HO-1 enzymatic activity results in the formation of the cytoprotective metabolites CO and biliverdin. In the skin, HO-1 is strongly induced after long wave ultraviolet radiation (UVA-1). Here we show that UVA-1 irradiation generates oxidized phospholipids derived from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) that mediate the expression of HO-1 in skin cells. Using EO6 antibodies that recognize oxidized phospholipids, we show that UVA-1 irradiation of dermal fibroblasts generates oxidation-specific epitopes. Irradiation of arachidonate-containing phospholipids with UVA-1 led to formation of defined lipid oxidation products including epoxyisoprostane-phosphatidylcholine that induced HO-1 expression in dermal fibroblasts, in keratinocytes, and in a three-dimensional epidermal equivalent model. In addition, we demonstrate that the oxidation of PAPC by UVA-1 is a singlet oxygen-dependent mechanism. Together, we present a novel mechanism of UVA-1-induced HO-1 expression that is mediated by the generation of biologically active phospholipid oxidation products. Because UVA-1 irradiation is a mainstay treatment of several inflammatory skin diseases, structural identification of UVA-1-generated biomolecules with HO-1-inducing capacity should lead to the development of drugs that could substitute for irradiation.

Protein C inhibitor (PCI) is a serpin with affinity for heparin and phosphatidylethanolamine (PE). We analyzed the interaction of PCI with different phospholipids and their oxidized forms. PCI bound to oxidized PE (OxPE), and oxidized and unoxidized phosphatidylserine (PS) immobilized on microtiter plates and in aqueous suspension. Binding to OxPE and PS was competed by heparin, but not by the aminophospholipid-binding protein annexin V or the PCI-binding lipid retinoic acid. PS and OxPE stimulated the inhibition of activated protein C (aPC) by PCI in a Ca++-dependent manner, indicating that binding of both, aPC (Ca++ dependent) and PCI (Ca++ independent), to phospholipids is necessary. A peptide corresponding to the heparin-binding site of PCI abolished the stimulatory effect of PS on aPC inhibition. No stimulatory effect of phospholipids on aPC inhibition was seen with a PCI mutant lacking the heparin-binding site. A heparin-like effect of phospholipids (OxPE) was not seen with antithrombin III, another heparin-binding serpin, suggesting that it is specific for PCI. PCI and annexin V were found to be endogenously colocalized in atherosclerotic plaques, supporting the hypothesis that exposure of oxidized PE and/or PS may be important for the local regulation of PCI activity in vivo.

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.

Oxidized phospholipids are generally recognized as deleterious factors involved in disease pathogenesis. This review summarizes the data suggesting that under certain biological conditions the opposite is correct, namely that OxPLs can also induce protective effects. Examples that are discussed in the review include upregulation of antioxidant genes, inhibition of inflammatory signaling pathways through Nrf2-dependent and -independent mechanisms, antagonism of Toll-like receptors, immuno-modulating and immuno-suppressive action of OxPLs in adaptive immunity and autoimmune disease, activation of PPARs known for their anti-inflammatory action, as well as protective action against lung edema in acute lung inflammation. The data support the notion that oxidation of phospholipids provides a negative feedback preventing damage to host tissues due to uncontrolled inflammation and oxidative stress.

DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell–driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase–meditated (12/15-LO–mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO–encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO–deficient DCs to 12/15-LO–derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO–deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune encephalomyelitis in mice lacking 12/15-LO resulted in an exacerbated Th17-driven autoimmune disease. Together, our data reveal that 12/15-LO controls maturation of DCs and implicate enzymatic lipid oxidation in shaping the adaptive immune response.

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2–generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.

Endothelial lipase (EL) is a phospholipase with little triacylglycerol lipase activity. To assess structural and functional properties of EL-HDL (EL-modified high-density lipoprotein), HDL was incubated with conditioned medium from Cos-7 cells infected with adenovirus encoding human EL. After re-isolation of HDL by ultracentrifugation, TLC and HPLC analyses revealed that EL-HDL was markedly depleted in phosphatidylcholine and enriched in lyso-phosphatidylcholine compared with LacZ-HDL (control HDL) incubated with conditioned medium from Cos-7 cells infected with adenovirus encoding β-galactosidase. The EL-HDL was enriched in non-esterified fatty acids and, as revealed by lipid electrophoresis, was more negatively charged than control HDL. The HDL particle size as well as the total cholesterol, free cholesterol and triacylglycerol content of HDL were not significantly altered after EL modification. The ability of EL-HDL to mediate 3H-cholesterol efflux from SR-BI (scavenger receptor B type I) overexpressing Chinese-hamster ovary cells was impaired and markedly lower compared with LacZ-HDL at HDL concentrations of 100 μg/ml and above. Studies with 125I-labelled HDL showed almost unaltered binding affinity (Km values) and a slightly but significantly decreased binding capacity (Bmax values) of EL-HDL to SR-BI, compared with LacZ-HDL. The ATP-binding-cassette transporter A1-dependent cholesterol and phospholipid effluxes were not affected by EL modification. From these results, we concluded that EL modification alters chemical composition and physical properties of HDL, resulting in its decreased binding capacity to SR-BI and a diminished ability to mediate SR-BI-dependent cholesterol efflux.

Short- and long-chain 1-O-alkyl-2-acylaminodeoxyglycero- and alkoxy-alkylphosphonic acid p-nitrophenyl esters were synthesized as inhibitors for analytical and mechanistic studies on lipolytic enzymes. The respective compounds contain perylene or nitrobenzoxadiazole as reporter fluorophores covalently bound to the omega-ends of the respective 2-acylamino- and alkoxy- residues. Their inhibitory effects on the activities of three selected lipases showing different substrate preferences were determined, including the lipases from Rhizopus oryzae, Pseudomonas species, and Pseudomonas cepacia. R. oryzae lipase reacted much better with the single-chain inhibitors than the two-chain deoxyglycerolipids. In contrast, P. cepacia lipase was inactivated by perylene-containing two-chain phosphonate (XXII) to a larger extent as compared to the other inhibitors whereas Pseudomonas species lipase interacted efficiently and without any preferences with all inhibitors used in this study. In summary, the different lipases show a very characteristic reactivity pattern not only with respect to triacylglycerol substrates but also to their structurally related inhibitors. Thus, the novel phosphonates might be useful tools not only for analysis and discrimination of known lipolytic enzymes but also for discovery of yet unknown lipases/esterases in biological samples.

Sec14p of the yeast Saccharomyces cerevisiae is involved in protein secretion and regulation of lipid synthesis and turnover in vivo, but acts as a phosphatidylinositol-phosphatidylcholine transfer protein in vitro. In this work, the five homologues of Sec14p, Sfh1p-Sfh5p, were subjected to biochemical and cell biological analysis to get a better view of their physiological role. We show that overexpression of SFH2 and SFH4 suppressed the sec14 growth defect in a more and SFH1 in a less efficient way, whereas overexpression of SFH3 and SFH5 did not complement sec14. Using C-terminal yEGFP fusions, Sfh2p, Sfh4p and Sfh5p are mainly localized to the cytosol and microsomes similar to Sec14p. Sfh1p was detected in the nucleus and Sfh3p in lipid particles and in microsomes. In contrast to Sec14p, which inhibits phospholipase D1 (Pld1p), overproduction of Sfh2p and Sfh4p resulted in the activation of Pld1p-mediated phosphatidylcholine turnover. Interestingly, Sec14p and the two homologues Sfh2p and Sfh4p downregulate phospholipase B1 (Plb1p)-mediated turnover of phosphatidylcholine in vivo. In summary, Sfh2p and Sfh4p are the Sec14p homologues with the most pronounced functional similarity to Sec14p, whereas the other Sfh proteins appear to be functionally less related to Sec14p.

Hypochlorous acid/hypochlorite (HOCl/OCl(-)), a potent oxidant generated in vivo by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, alters the physiological properties of high density lipoprotein (HDL) by generating a proatherogenic lipoprotein particle. On endothelial cells lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) and scavenger receptor class B, type I (SR-BI), act in concert by mediating the holoparticle of and selective cholesteryl ester uptake from HOCl-HDL. We therefore investigated the ligand specificity of HOCl-HDL to SR-BI-overexpressing Chinese hamster ovary cells. Binding of HOCl-HDL was saturable, and the degree of HOCl modification was the determining factor for increased binding affinity to SR-BI. Competition experiments further confirmed that HOCl-HDL binds with increased affinity to the same or overlapping domain(s) of SR-BI as does native HDL. Furthermore, SR-BI-mediated selective HDL-cholesteryl ester association as well as time- and concentration-dependent cholesterol efflux from SR-BI overexpressing Chinese hamster ovary cells were, depending on the degree of HOCl modification of HDL, markedly impaired. The most significant findings of this study were that the presence of very low concentrations of HOCl-HDL severely impaired SR-BI-mediated bidirectional cholesterol flux mediated by native HDL. The colocalization of immunoreactive HOCl-modified epitopes with apolipoprotein A-I along with deposits of lipids in serial sections of human atheroma shown here indicates that the myeloperoxidase-H(2)O(2)-halide system contributes to oxidative damage of HDL in vivo.

Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1(-/-) mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections.

Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms.

Oxidized phospholipids (OxPLs) are increasingly recognized as signaling mediators that are not only markers of oxidative stress but are also "makers" of pathology relevant to disease pathogenesis. Understanding the biological role of individual molecular species of OxPLs requires the knowledge of their concentration kinetics in cells and tissues. In this work, we describe a straightforward "fingerprinting" procedure for analysis of a broad spectrum of molecular species generated by oxidation of the four most abundant species of polyunsaturated phosphatidylcholines (OxPCs). The approach is based on liquid-liquid extraction followed by reversed-phase HPLC coupled to electrospray ionization MS/MS. More than 500 peaks corresponding in retention properties to polar and oxidized PCs were detected within 8 min at 99 m/z precursor values using a single diagnostic product ion in extracts from human dermal fibroblasts. Two hundred seventeen of these peaks were fluence-dependently and statistically significantly increased upon exposure of cells to UVA irradiation, suggesting that these are genuine oxidized or oxidatively fragmented species. This method of semitargeted lipidomic analysis may serve as a simple first step for characterization of specific "signatures" of OxPCs produced by different types of oxidative stress in order to select the most informative peaks for identification of their molecular structure and biological role.

Objective Atherosclerotic lesions contain high concentrations of oxidized phospholipids (OxPLs) known to induce VEGF via the ATF4 arm of unfolded protein response (UPR), and to promote angiogenic reactions thus potentially contributing to the progression and destabilization of atherosclerotic plaques. In order to get further insights into the mechanisms of cellular stress-induced angiogenesis we studied the role of a specific microRNA (miR-663) in the mechanisms of VEGF induction by OxPLs and inducers of UPR. Methods miRNA and mRNA levels were determined using microarray profiling and qRT-PCR methods. Proteins were analyzed by Western blotting. miR-663 levels were changed by transfecting cells with synthetic oligonucleotides. Results OxPAPC elevated miR-663 in two types of human endothelial cells (ECs). Knockdown of miR-663 inhibited upregulation of VEGF mRNA in ECs treated by OxPAPC, OxPAPS or OxPAPA. In addition, silencing of miR-663 suppressed upregulation by OxPAPC of ATF4 mRNA and protein, as well as a downstream gene TRIB. Similarly to the inhibition of OxPAPC effects, knockdown of miR-663 suppressed elevation of ATF4, VEGF and TRIB in response to another inducer of UPR, tunicamycin. Overexpression of miR-663 reversed the inhibition of VEGF induction by miR-663 inhibitor. Conclusion miR-663 is critically important for 2 key events induced in ECs by stress agents and oxidized lipids, namely induction of transcription factor ATF4 and its downstream gene VEGF. These findings allow hypothesizing that miR-663 plays a general role in control of the ATF4 branch of UPR induced by different agents.

The generation of phospholipid oxidation products in atherosclerosis, sepsis, and lung pathologies affects endothelial barrier function, which exerts significant consequences on disease outcomes in general. Our group previously showed that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (OxPAPC) at low concentrations increases endothelial cell (EC) barrier function, but decreases it at higher concentrations. In this study, we determined the mechanisms responsible for the pulmonary endothelial cell barrier dysfunction induced by high OxPAPC concentrations. OxPAPC at a range of 5–20 μg/ml enhanced EC barriers, as indicated by increased transendothelial electrical resistance. In contrast, higher OxPAPC concentrations (50–100 μg/ml) rapidly increased EC permeability, which was accompanied by increased total cell protein tyrosine (Tyr) phosphorylation, phosphorylation at Tyr-418, the activation of Src kinase, and the phosphorylation of adherens junction (AJ) protein vascular endothelial cadherin (VE-cadherin) at Tyr-731 and Tyr-658, which was not observed in ECs stimulated with low OxPAPC doses. The early tyrosine phosphorylation of VE-cadherin was linked to the dissociation of VE-cadherin–p120-catenin/β-catenin complexes and VE-cadherin internalization, whereas low OxPAPC doses promoted the formation of VE-cadherin–p120-catenin/β-catenin complexes. High but not low doses of OxPAPC increased the production of reactive oxygen species (ROS) and protein oxidation. The inhibition of Src by PP2 and ROS production by N-acetyl cysteine inhibited the disassembly of VE-cadherin–p120-catenin complexes, and attenuated high OxPAPC-induced EC barrier disruption. These results show the differential effects of OxPAPC doses on VE-cadherin–p120-catenin complex assembly and EC barrier function. These data suggest that the rapid tyrosine phosphorylation of VE-cadherin and other potential targets mediated by Src and ROS-dependent mechanisms plays a key role in the dissociation of AJ complexes and EC barrier dysfunction induced by high OxPAPC doses.

The coordinated and non-inflammatory phagocytosis of apoptotic cells is crucial to maintain immunological tolerance. During inflammation, however, ingestion of apoptotic material by inflammatory phagocytes can provoke a break in self-tolerance. Hence, and though poorly understood, mechanisms governing the sorting of apoptotic cells into distinct and differentially-activated subsets of phagocytes are essential to prevent autoimmunity. Here we identify the enzyme 12/15-lipoxygenase (12/15-LO) as a crucial factor orchestrating the clearance of apoptotic cells under inflammatory conditions. During peritonitis, the ingestion of apoptotic cells is confined to a distinct population of alternatively-activated, 12/15-LO-expressing resident macrophages (resMΦ). Deletion of 12/15-LO changed this pattern and neighbouring inflammatory macrophages (infMΦ) start to engulf apoptotic cells. We hypothesized that resMΦ exert a paracrine and 12/15-LO-mediated inhibitory activity on the phagocytic capacity of infMΦ and identified oxidation products of phosphatidyl ethanolamine (oxPE) as the corresponding mediators. oxPE is generated by the action of 12/15-LO in resMΦ and consequently exposed on the macrophages' surfaces. Here, oxPE binds, blocks and scavenges soluble MFG-E8 protein and thereby selectively blocks the major pathway involved in the uptake of apoptotic cells into infMΦ. In turn, we observed a break in self-tolerance and lupus-like autoimmune disease in aged 12/15-LO-deficient mice. These mice displayed spontaneous production of autoantibodies and glomerulonephritis, which both exacerbated after apoptotic challenge in the pristane-induced model of experimental murine lupus. Our data point towards a so far unrecognized role for enzymatic lipid oxidation during the maintenance of self-tolerance and identify a mechanism, which orchestrates the cell- and context-specific uptake of antigens by different subsets of phagocytes, imposing a new paradigm in our understanding of the clearance of apoptotic cells.

Besides their well-characterized proinflammatory and proatherogenic effects, oxidized phospholipids, such as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-phosphocholine) have been shown to have beneficial responses in vascular cells via induction of antioxidant enzymes such as heme oxygenase-1. We therefore hypothesized that oxPAPC could evoke a general cytoprotective response via activation of antioxidative transcription factor Nrf2. Here, we show that oxPAPC increases nuclear accumulation of Nrf2. Using the small interfering RNA approach, we demonstrate that Nrf2 is critical in mediating the induction of glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase-1 (NQO1) by oxPAPC in human endothelial cells, whereas the contribution to the induction of heme oxygenase-1 was less significant. The induction of GCLM and NQO1 was attenuated by reduction of electrophilic groups with sodium borohydrate, as well as treatment with thiol antioxidant N -acetylcysteine, suggesting that the thiol reactivity of oxPAPC is largely mediating its effect on Nrf2-responsive genes. Moreover, we show that oxidized phospholipid having a highly electrophilic isoprostane ring in its sn-2 position is a potent inducer of Nrf2 target genes. Finally, we demonstrate that the oxPAPC-inducible expression of heme oxygenase-1, GCLM, and NQO1 is lower in Nrf2-null than wild-type mouse carotid arteries in vivo. We suggest that the activation of Nrf2 by oxidized phospholipids provides a mechanism by which their deleterious effects are limited in the vasculature.