Oxidation Of Membrane Phospholipids Research Articles

Abstract 3093: Can Inhibiting Ferroptosis And Subsequent Endothelial Glycocalyx Shedding Reduce The Morbidity And Mortality Of Hemorrhagic Shock?

Background: Trauma patients with hemorrhagic shock accounts for 40% of preventable death. Shock-induced endotheliopathy has garnered interest recently and one aspect of this, endothelial glycocalyx shedding, has shown to promote coagulopathy following hemorrhage. Ferroptosis is an iron dependent, non-apoptotic form of controlled cell death triggered by the oxidation of membrane phospholipids. Liproxstatin-1 (LPX1) is an inhibitor of this process and can protect against oxidative damage to the endothelial cell membrane and subsequent shedding of glycocalyx. We hypothesized that LPX1 would reduce endothelial glycocalyx damage in hemorrhagic shock. Methods: A rat model of hemorrhagic shock and resuscitation was used to assess glycocalyx disruption in the lungs via fluorescent-labeled wheat germ agglutinin staining in frozen tissue and by Syndecan-1 ELISA on plasma samples. A control group (n=5) resuscitated only with Lactated Ringer’s solution, following hemorrhage and resuscitation (H/R) was compared against an experimental group (n=5) that received LPX1 following hemorrhage and before resuscitation. Results: Glycocalyx shedding (assessed by an increase in plasma Syndecan-1 levels) was prevented in the LPX1 treated group compared to control group. Lung tissue staining of the glycocalyx also showed a greater glycocalyx level in the LPX1 group than in the control (40.4 vs 36.3 arbitrary units). Additionally, LPX1 treated animals were able to sustain a higher mean arterial pressure (82 vs 45mmHg), and oxygen saturation throughout the resuscitation period measured at 15 and 30 minutes following LPX1 infusion (98.4% SpO2 vs. 99.0% SpO2 at 30 minutes resuscitation). Conclusions: Ferroptosis inhibitor LPX1 can reduce morbidity acutely during hemorrhagic shock and can protect the endothelial cell glycocalyx from oxidative damage due to its ability to slow the generation of reactive oxygen species and lipid hydroperoxides.

Nanoactuator for Neuronal Optoporation.

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing "on-demand" light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation.

Hydrogen sulfide guards myoblasts from ferroptosis by inhibiting ALOX12 acetylation

Recognized as a novel and important gasotransmitter, hydrogen sulfide (H2S) is widely present in various tissues and organs. Cystathionine gamma-lyase (CSE)-derived H2S has been shown to regulate oxidative stress and lipid metabolism. The aim of the present study is to examine the role of H2S in ferroptosis and lipid peroxidation in mouse myoblasts and skeletal muscles. Ferroptosis agonist RSL3 inhibited the expressions of Gpx4 and reduced CSE/H2S signaling, which lead to increased oxidative stress, lipid peroxidation, and ferroptotic cell death. In addition, ferroptosis antagonist ferrostatin-1 (Fer-1) up-regulated the expression of CSE, scavenged the generation of reactive oxygen species (ROS) and lipid peroxidation, and improved cell viability. Exogenously applied NaHS was also able to block RSL3-induced ferroptotic cell death. Neither RSL3 nor H2S affected cell apoptosis. Furthermore, H2S reversed RSL3-induced Drp1 expression and mitochondrial damage, which lead to abnormal lipid metabolism as evidenced by altered expressions of ACSL4, FAS, ACC and CPT1 as well as higher acetyl-CoA contents in both cytoplasm and mitochondria. RSL3 promoted the protein expression and acetylation of ALOX12, a key protein in initiating membrane phospholipid oxidation, while the addition of NaHS attenuated ALOX12 acetylation and protected from membrane lipid peroxidation. Moreover, we observed that CSE deficiency alters the expressions of ferroptosis and lipid peroxidation-related proteins and enhances global protein acetylation in mouse skeletal muscles under aging or injury conditions. These results indicate that downregulation of CSE/H2S signaling would contribute to mitochondrial damage, abnormal lipid metabolism, membrane lipid peroxidation, and ferroptotic cell death. CSE/H2S system can be a target for preventing ferroptosis in skeletal muscle.

Ozonation of three different fungal conidia associated with apple disease: Importance of spore surface and membrane phospholipid oxidation.

Although ozone (O3) is a well‐known bactericide and fungicide, the required dose of ozone can depend significantly on the targeted pathogens. The present research evaluates the variation in sensibility to ozone of three fungal species from a single fungal group. The three fungal species selected, Venturia inaequalis, Botrytis cinerea, and Neofabreae alba, belong to the Ascomycota group and attack apples. The fungi were exposed to ozone by bubbling directly into the spore solutions (treatment period ranged from 0.5 to 4 min, ozone concentration in inlet gas ranged from 1 to 30 g/m3). The rates of germination were determined, and the level of peroxidation of the lipid membrane was quantified based on the malondialdehyde (MDA) production. The results indicate that ozone effectively reduces spore development and suggest that the fungi differ in sensitivity. To reduce by 50% the spore germination rate of N. alba, B. cinerea, and V. inaequalis requires ozone doses of 0.01, 0.03, and 0.07 mg/ml, respectively. Spore sensitivity seems to be directly linked to spore surface. For all the fungal species, the MDA level and the level of spore inactivation both increase with ozone dose, which confirms that ozone alters the cell membrane.

Platelet-Activating Factor-Induced Reduction in Contact Hypersensitivity Responses Is Mediated by Mast Cells via Cyclooxygenase-2-Dependent Mechanisms.

Platelet-activating factor (PAF) stimulates numerous cell types via activation of the G protein-coupled PAF receptor (PAFR). PAFR activation not only induces acute proinflammatory responses, but it also induces delayed systemic immunosuppressive effects by modulating host immunity. Although enzymatic synthesis and degradation of PAF are tightly regulated, oxidative stressors, such as UVB, chemotherapy, and cigarette smoke, can generate PAF and PAF-like molecules in an unregulated fashion via the oxidation of membrane phospholipids. Recent studies have demonstrated the relevance of the mast cell (MC) PAFR in PAFR-induced systemic immunosuppression. The current study was designed to determine the exact mechanisms and mediators involved in MC PAFR-mediated systemic immunosuppression. By using a contact hypersensitivity model, the MC PAFR was not only found to be necessary, but also sufficient to mediate the immunosuppressive effects of systemic PAF. Furthermore, activation of the MC PAFR induces MC-derived histamine and PGE2 release. Importantly, PAFR-mediated systemic immunosuppression was defective in mice that lacked MCs, or in MC-deficient mice transplanted with histidine decarboxylase- or cyclooxygenase-2-deficient MCs. Lastly, it was found that PGs could modulate MC migration to draining lymph nodes. These results support the hypothesis that MC PAFR activation promotes the immunosuppressive effects of PAF in part through histamine- and PGE2-dependent mechanisms.

Low levels of IgM antibodies recognizing oxidation-specific epitopes are associated with human non-alcoholic fatty liver disease

BackgroundLipid oxidation of membrane phospholipids is accompanied by the formation of oxidation-specific epitopes (OSE). These epitopes are recognized by specific antibodies and represent danger-associated molecular patterns that are generated during chronic inflammatory processes. In a murine model for hepatic inflammation during non-alcoholic fatty liver disease (NAFLD), increased antibody levels targeting OSE were found to be protective. Here, our aim was to determine an association between OSE-specific antibody titers and NAFLD in humans.MethodsIgM and IgG levels with specificity for various OSE were assessed in the plasma of patients with NAFLD (n = 71) and healthy controls (n = 68). Antibody titers were comprehensively analyzed in patients with NAFLD after classification by histological evaluation of liver biopsies. Statistical analysis was performed to determine significant correlations and odds ratios. To study the specificity for NAFLD, plasma antibody titers were measured in patients with hepatitis C (n = 40) and inflammatory bowel disease (n = 62).ResultsIgM titers against OSE were lower in patients with NAFLD compared to controls. Further biopsy-based classification of patients with NAFLD did not show any difference in IgM levels. Plasma IgM titers towards the P1 mimotope demonstrated an inverse correlation with markers for obesity, systemic inflammation, and liver damage. In contrast, hepatitis C and increased disease activity during inflammatory bowel disease was not associated with reduced IgM titers.ConclusionsOur data highlight the importance of immune recognition of OSE by IgM antibodies in the pathophysiology of NAFLD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-016-0652-0) contains supplementary material, which is available to authorized users.

Receptor-Mediated Mechanism Controlling Tissue Levels of Bioactive Lipid Oxidation Products

Oxidative stress is an important contributing factor in several human pathologies ranging from atherosclerosis to cancer progression; however, the mechanisms underlying tissue protection from oxidation products are poorly understood. Oxidation of membrane phospholipids, containing the polyunsaturated fatty acid docosahexaenoic acid, results in the accumulation of an end product, 2-(ω-carboxyethyl)pyrrole (CEP), which was shown to have proangiogenic and proinflammatory functions. Although CEP is continuously accumulated during chronic processes, such as tumor progression and atherosclerosis, its level during wound healing return to normal when the wound is healed, suggesting the existence of a specific clearance mechanism. To identify the cellular and molecular mechanism for CEP clearance. Here, we show that macrophages are able to bind, scavenge, and metabolize carboxyethylpyrrole derivatives of proteins but not structurally similar ethylpyrrole derivatives, demonstrating the high specificity of the process. F4/80(hi) and M2-skewed macrophages are much more efficient at CEP binding and scavenging compared with F4/80(lo) and M1-skewed macrophages. Depletion of macrophages leads to increased CEP accumulation in vivo. CEP binding and clearance are dependent on 2 receptors expressed by macrophages, CD36 and toll-like receptor 2. Although knockout of each individual receptor results in diminished CEP clearance, the lack of both receptors almost completely abrogates macrophages' ability to scavenge CEP derivatives of proteins. Our study demonstrates the mechanisms of recognition, scavenging, and clearance of pathophysiologically active products of lipid oxidation in vivo, thereby contributing to tissue protection against products of oxidative stress.

Abstract W P411: Effect of Normobaric Oxygen Therapy (NBO) on Oxidative Stress in Acute Ischemic Stroke

BACKGROUND: There is little human data concerning the effect of hyperoxia on markers of oxidative stress after acute ischemic stroke (AIS); rodent studies show conflicting results. METHODS: In this NIH-SPOTRIAS Trial of normobaric oxygen therapy (NBO) in AIS, tPA-ineligible subjects with imaging-confirmed AIS < 9 hours and NIHSS score 4 or higher were randomized to NBO or Medical Air, delivered for 8 hrs. Of n=85 enrolled, we analyzed n=53 (24 NBO, 29 Air) who had NIHSS, oxidative stress biomarkers, and serial CT/MRI obtained at 0 hrs (admission/pre-therapy), 4 hrs (during therapy) and 48 hrs (post-therapy). RESULTS: At baseline, the NBO arm showed a trend for higher DWI lesion volumes (39.8±42 vs 20.3±20 cc, p=0.06), but there was no significant difference between NBO and Air for mean age (73±15y vs 74±14y, p=0.9), median NIHSS (11.5 vs. 9.0, p=0.5), or perfusion-MRI "mean transit time" lesion volumes (100.9 vs 74.3 cc, p=0.6). Pre-therapy antioxidant capacity, assessed with the oxygen radical absorbance capacity assay, was significantly lower in the NBO arm (p=0.02), suggestive of greater baseline oxidative stress associated with larger infarct volumes. Pre-therapy levels of the matrix metalloproteinases MMP-2 and MMP-9, and F2isoProstane (F2isoP, a direct marker of non-enzymatic oxidation of membrane phospholipids) were similar between groups. There was no significant change from 0-4 hours or from 0-48 hours in NIHSS scores, DWI volumes, and levels of MMP-2, MMP-9, and F2isoP (Table). The NBO and Air groups showed no significant differences in rates of tissue reperfusion, or indirect MRI markers of free radical injury such as stroke-related mass effect, brain hemorrhage, or the hyperacute reperfusion injury marker (HARM) sign. CONCLUSION: In patients with acute ischemic stroke, there is no significant change in direct and indirect markers of oxidative stress with hyperoxia therapy.

Characterization of cholesterol crystalline domains in model and biological membranes using X-ray diffraction.

In this review, we discuss the use of small-angle X-ray diffraction approaches in studying the formation of cholesterol crystalline domains in membranes derived from model and biological sources. Numerous studies have shown that monomeric cholesterol can self-associate and form immiscible, membrane-restricted domains as a result of increased membrane concentration or systematic oxidation of membrane phospholipids. These domains are observed, in an X-ray diffraction pattern, as reflections describing a unit cell periodicity of 34 Å, which is consistent with cholesterol molecules arranged in a tail-to-tail, bilayer conformation. In vascular smooth muscle cells isolated from animal models of atherosclerosis, plasmalemmal cholesterol domain formation is associated with cellular dysfunction, including the disruption of calcium transport mechanisms. Cholesterol domains are also observed in macrophages and precede the deposition of extracellular cholesterol crystals in the atheroma. We have also shown that cholesterol domains can be produced in model membranes following exposure to oxidative stress and other disease-like conditions such as hyperglycemia. By contrast, cholesterol domains appear to be essential to the normal physiology of certain cell groups such as those of the human ocular lens. Cholesterol domains are a prominent structural feature of the lens fiber cell plasma membrane where they are believed to facilitate lens tissue transparency and interfere with various mechanisms of cataract formation. These contrasting biologic effects highlight the complex role that cholesterol domains play in cell plasma membrane structure-function relationships in both health and disease.KeywordsCholesterol DomainsFiber Cell Plasma MembraneFiber CellsCalcium Transport MechanismsTulenkoThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

SP0051 Activated Immune Cells Enzymatically Oxidize Membrane Lipids on Activation

Several new families of bioactive lipids were identified that form through the enzymatic oxidation of membrane phospholipids in circulating innate immune cells and platelets. These comprise eicosanoids attached to phosphatidylethanolamine...

Cancellation of cellular responses to nanoelectroporation by reversing the stimulus polarity.

Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca(2+) mobilization and cell survival with those of bipolar 60+60 and 300+300ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1-60), and amplitudes (15-60kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-µs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.

Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.

Circulation Research Thematic Synopsis Diabetes and Obesity

<i>Circulation Research</i> Thematic Synopsis Diabetes and Obesity

Photodynamic oxidation of Staphylococcus warneri membrane phospholipids: new insights based on lipidomics

The photodynamic process involves the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of oxidizing biological molecules, such as lipids. However, the effect of the photodynamic process in the bacterial phospholipid profile by a photosensitizer has never been reported. A lipidomic approach was used to study the photodynamic oxidation of membrane phospholipids of Staphylococcus warneri by a tricationic porphyrin [5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide, Tri-Py(+)-Me-PF]. S. warneri (10(8) colony forming units mL(-1)) was irradiated with white light (4 mW cm(-2), 21.6 J cm(-2)) in the presence of Tri-Py(+)-Me-PF (5.0 μM). Non-photosensitized bacteria were used as control (irradiated without porphyrin). After irradiation, total lipids were extracted and separated by thin-layer chromatography (TLC). Isolated fractions of lipid classes were quantified by phosphorus assay and analyzed by mass spectrometry (MS): off-line TLC/ESI-MS, hydrophilic interaction (HILIC)-LC/MS and MS/MS. The most representative classes of S. warneri phospholipids were identified as phosphatidylglycerols (PGs) and cardiolipins (CLs). Lysyl-phosphatidylglycerols (LPGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs) and phosphatidic acids (PAs) were also identified. After photodynamic treatment, an overall increase in the relative abundance of PGs was observed as well as the appearance of new oxidized species from CLs, including hydroxy and hydroperoxy derivatives. Formation of high amounts of lipid hydroperoxides was confirmed by FOX2 assay. Photodynamic oxidation of phospholipid standards revealed the formation of hydroperoxy and dihydroperoxy derivatives, confirming the observed CL oxidized species in S. warneri. Membrane phospholipids of S. warneri are molecular targets of the photoinactivation process induced by Tri-Py(+) -Me-PF. The overall modification in the relative amount of phospholipids and the formation of lipid hydroxides and hydroperoxides indicate the lethal damage caused to photosensitized bacterial cells.

Peptidyl-prolyl cis/trans-Isomerase A1 (Pin1) Is a Target for Modification by Lipid Electrophiles

Oxidation of membrane phospholipids is associated with inflammation, neurodegenerative disease, and cancer. Oxyradical damage to phospholipids results in the production of reactive aldehydes that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme that catalyzes the conversion of the peptide bond of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael adducts at the active site residues His-157 and Cys-113. Time and concentration dependencies indicate that Cys-113 is the primary site of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and Western blotting with anti-Pin1 antibody. Furthermore, orbitrap MS data support the adduction of Cys-113 in the Pin1 active site upon HNE treatment of MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.

Structural Identification and Cardiovascular Activities of Oxidized Phospholipids

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.

Abstract 3622: Oxidized LDL-derived Reactive Aldehydes Induce ER Stress and Trigger the Unfolded Protein Response: Implications for Atherosclerotic Lesion Formation

All stages of atherosclerotic lesion formation are associated with the accumulation of proteins modified by aldehydes that are generated from the oxidation of membrane phospholipids and lipoproteins. Although aldehyde-adducted proteins elicit strong immunogenic responses, the role of lipid peroxidation products in promoting and sustaining atherogenesis remains unclear. To examine aldehyde-induced protein modification and to assess its contribution to vascular stress, vascular smooth muscle and endothelial cells (EC) were exposed to 4-hydroxy-trans-2-nonenal (HNE), one of the most abundant and reactive oxidized lipid-derived aldehyde (LDA). Exposure to HNE (25 μM, 2h) resulted in extensive modification of cellular proteins in both cell types. Majority of these proteins, identified by MALDI/MS analysis, were ER-resident protein chaperones that assist in protein folding. Confocal imaging of HNE treated EC showed that the modified proteins were associated with the ER. Exposure to HNE led to the splicing of the bZIP transcription factor, XBP-1, a characteristic feature of the alarm phase of ER stress, and an increase in the phosphorylation of eIF2α, which regulates the expression of multiple ER-stress dependent transcription factors. These transcription factors, ATF3, ATF4, and CHOP were also strongly (2–5-fold) induced by HNE. Treatment with HNE was associated with a 2–5 fold increase in the expression of ER chaperones, GRP78 and HERP, indicating that HNE triggers the adaptive phase of the response, which increases the protein folding capacity of the ER. Pretreatment with the chemical chaperone, phenylbutyric acid (PBA), to facilitate protein folding, completely abolished HNE-induced increase in the expression of ATF3. EC treated with HNE enhanced monocyte adhesion, which was attenuated by PBA, indicating that early stages of lesion formation may be ER-stress dependent. In intermediate arterial lesion of 20 week old apoE-null mice, HNE-adducted proteins were localized to areas immunopositive for ATF3, ATF4, and phospho-PERK. These results suggest that protein modifications by LDA induce ER stress and the alarm and adaptive responses to this stress may be significant determinants of lesion progression and stability.

Effect of Low pH on the Susceptibility of Isolated Cod (Gadus morhua) Microsomes to Lipid Oxidation

During the extraction of muscle to produce protein isolates by acid or alkali solubilization, membranes are exposed to abnormally low or high pH. Low but not high pH treatment induces rapid oxidation of membrane phospholipids in the presence of hemoglobin. The goal of this research work was to study the oxidative stability of microsomes under the conditions met during acid solubilization. Isolated microsomes from cod muscle were used as a model system. At pH 5.3 or lower, 99% of isolated cod membranes sedimented at low centrifugation speeds. Isolated membranes that were exposed to pH 3.0 were less susceptible to hemoglobin-mediated lipid oxidation. Cod hemoglobin exposed to pH 3 was rendered less pro-oxidative than the untreated cod hemoglobin. However, when microsomes and hemoglobin were together exposed to low pH, oxidation was promoted. Citric acid and calcium chloride, as well as press juice isolated from cod muscle, were able to inhibit lipid oxidation of microsomal suspensions.

Apoptotic cells as sources for biologically active oxidized phospholipids.

Acute inflammation is characterized by an accumulation of polymorphonuclear cells (PMNs), generation of reactive oxygen species, subsequent apoptosis of PMNs, and finally phagocytosis of apoptotic cells by macrophages. Recently, it has been demonstrated that during apoptosis oxidation of membrane phospholipids, especially phosphatidylserine, occurs. Moreover, we have shown that membrane vesicles released from apoptotic cells contain biologically active oxidized phospholipids. The involvement of oxidized phospholipids in the development of atherosclerosis, which is described as a chronic inflammatory disease, is increasingly recognized. These oxidized phospholipids were shown to induce several proinflammatory genes, such as monocyte chemoattractant protein 1 or interleukin-8, and it is hypothesized that lipid oxidation products also play a role in other chronic inflammatory disorders. On the other hand, oxidized phospholipids were shown to exert antiendotoxin effects by inhibiting lipopolysaccharide-induced signaling, representing a possible feedback loop during gram-negative infection. Additionally, it has been described that oxidized phospholipids are capable of inducing genes such as heme oxygenase-1 that are important for the resolution of acute inflammation. Moreover, oxidized phospholipids serve as recognition signals on apoptotic cells facilitating phagocytosis. In this review, we discuss the hypothesis that oxidized phospholipids generated in apoptotic cells (a) propagate chronic inflammation and (b) contribute to the resolution of acute inflammation.

Lipid Oxidation Enhances the Function of Activated Protein C

Although lipid oxidation products are usually associated with tissue injury, it is now recognized that they can also contribute to cell activation and elicit anti-inflammatory lipid mediators. In this study, we report that membrane phospholipid oxidation can modulate the hemostatic balance. Oxidation of natural phospholipids results in an increased ability of the membrane surface to support the function of the natural anticoagulant, activated protein C (APC), without significantly altering the ability to support thrombin generation. Lipid oxidation also potentiated the ability of protein S to enhance APC-mediated factor Va inactivation. Phosphatidylethanolamine, phosphatidylserine, and polyunsaturation of the fatty acids were all required for the oxidation-dependent enhancement of APC function. A subgroup of thrombotic patients with anti-phospholipid antibodies specifically blocked the oxidation-dependent enhancement of APC function. Since leukocytes are recruited and activated at the thrombus or sites of vessel injury, our findings suggest that after the initial thrombus formation, lipid oxidation can remodel the membrane surface resulting in increased anticoagulant function, thereby reducing the thrombogenicity of the thrombus or injured vessel surface. Anti-phospholipid antibodies that block this process would therefore be expected to contribute to thrombus growth and disease.