This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huber, J. Articles by Leitinger, N. Search for Related Content PubMed PubMed Citation Articles by Huber, J. Articles by Leitinger, N. Related Collections Apoptosis Pathophysiology Risk Factors Lipid and lipoprotein metabolism Mechanism of atherosclerosis/growth factors

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:101.)
© 2002 American Heart Association, Inc.

Vascular Biology

Oxidized Membrane Vesicles and Blebs From Apoptotic Cells Contain Biologically Active Oxidized Phospholipids That Induce Monocyte-Endothelial Interactions

Joakim Huber; Anja Vales; Goran Mitulovic; Michael Blumer; Rainer Schmid; Joseph L. Witztum; Bernd R. Binder; Norbert Leitinger

From the Department of Vascular Biology and Thrombosis Research (J.H., A.V., B.R.B., N.L.), University of Vienna; the Clinical Institute of Medical and Chemical Laboratory Analysis (G.M., R.S.), General Hospital Vienna; and the Department of Zoology (M.B.), University of Vienna, Vienna, Austria; and the Department of Medicine (J.L.W.), University of California, San Diego.

Correspondence to Dr Norbert Leitinger, Department of Vascular Biology and Thrombosis Research, University of Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria. E-mail norbert.leitinger{at}univie.ac.at

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Membrane vesicles (MVs) released from activated cells and blebs from apoptotic cells are increased in patients with vascular disease and in those with atherosclerotic lesions, and their contribution to inflammatory reactions has been suggested. At sites of inflammation, MVs could serve as rapidly available substrates for peroxidation, carry oxidized compounds to activate other cells, and amplify inflammation. Here, we show that MVs released from tert-butyl hydroperoxide–treated endothelial cells (ECs) and apoptotic blebs, but not MVs from Ca²⁺ ionophore–treated ECs, stimulate monocyte adhesion to ECs, an important step in atherogenesis. We show that oxidized phospholipids, such as the previously identified 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), are responsible for biological activity in MVs and apoptotic blebs. Natural antibodies from apolipoprotein E–null mice that recognize POVPC also recognize oxidized MVs, and pretreatment of MVs with these antibodies inhibits their ability to activate ECs. Furthermore, the biological activity of oxidized MVs is inhibited by platelet-activating factor receptor antagonists, which have been shown to inhibit the action of POVPC. Taken together, we show that oxidized MVs and apoptotic blebs stimulate ECs to specifically bind monocytes, with oxidized phospholipids (POVPC) being the active principle. In addition to oxidized lipoproteins, oxidized MVs and apoptotic blebs may play an important role in chronic inflammatory diseases, such as atherosclerosis.

Key Words: membrane vesicles • apoptotic blebs • oxidized phospholipids • endothelial cells • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Activation of vascular cells or circulating blood cells by various agonists such as Ca²⁺ ionophore, terminal complement proteins C5b-9, thrombin, lipopolysaccharide, tumor necrosis factor (TNF)-, or hydroperoxide^1–6 causes the release of parts of the cell membrane (membrane vesicles [MVs]), which is due to stimulation of different mechanisms, including increase in intracellular Ca²⁺ and calpain activation.⁵ In addition, cells undergoing apoptosis also shed parts of their membranes (apoptotic blebs),^7,8 and this process has been shown to be accompanied by oxidative stress.⁹

Modification of MVs is thought to render them biologically active, and there is increasing evidence that lipids in MVs can elicit cellular responses: it has been shown that after treatment with secretory phospholipase A₂, platelet-derived MVs activate endothelial cells (ECs) and cause platelet aggregation, with arachidonic acid being the active principle^10,11; MVs derived from platelets or red blood cells and treated with a combination of phospholipase A₂ and sphingomyelinase cause platelet aggregation, with lysophosphatidic acid being the active principle²; and ECs exposed to peroxides release MVs that contain oxidized phospholipids and activated neutrophils.⁴ These studies also indicate that modified MVs may play an important role in inflammation.^2,4,11–13 Indeed, levels of MVs were increased in patients with acute coronary syndrome,¹⁴ diabetes mellitus,¹⁵ and cardiopulmonary bypass,¹⁶ in human atherosclerotic plaques,¹⁷ and in pathological settings in which platelet activation was involved.^18–20 Particularly, EC-derived MV levels were increased in the peripheral blood of patients with coagulation abnormality³ and in patients with acute coronary syndrome.¹⁴ In addition, apoptosis was shown to play an important role in various pathological settings, such as cardiomyopathy, acute myocardial infarction, myocarditis, and atherosclerosis (see reviews^8,21), and high levels of apoptotic blebs were detected in atherosclerotic plaques.¹⁷

In the present study, we examined whether modified MVs and apoptotic blebs stimulate ECs to bind monocytes, an initiating event in atherogenesis. We demonstrate that apoptotic blebs and oxidatively modified, but not native, MVs contain proinflammatory oxidized phospholipids that elicit specific responses in ECs, leading to the adhesion of monocytes. Thus, oxidized MVs and apoptotic blebs may play an important role in atherogenesis and other chronic inflammatory diseases.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), dimyristoylphosphatidylcholine, A23187 (Ca²⁺ ionophore), E-Toxate (Limulus amoebocyte lysate), butylated hydroxytoluene (BHT), and polymyxin B were purchased from Sigma Chemical Co; tert-butyl hydroperoxide (t-BuOOH) was purchased from Aldrich. Platelet-activating factor (PAF) receptor antagonists Ginkolide B and rac-3-(N-octadecylcarbamoyl)-2-methoxy)-propyl-(2-thiazolioethyl)-phosphate (BN-52021 and CV-3988, respectively) were from BIOMOL. TNF- was from Boehringer-Mannheim. Monoclonal anti-human mouse E-selectin IgG antibody was from R&D Systems; goat anti-mouse IgG was from Accurate Chemical & Scientific Corp; biotinylated goat anti-mouse IgM was from Accurate Chemical & Scientific Corp; monoclonal IgM against tissue plasminogen activator was from Technoclone; the ApoAlert annexin V apoptosis kit was from Clontech; and monoclonals E03, E06, and E014 (all IgMs) were from the La Jolla Specialized Center of Research core immunology laboratory.

Phospholipid Preparation
Oxidized PAPC (Ox-PAPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) were prepared from PAPC as described previously.^22,23

Tissue Culture
Human umbilical vein ECs were prepared and cultured as described previously²⁴ and used for experiments at passages 2 to 5.

Isolation of MVs and Apoptotic Blebs
ECs were exposed to t-BuOOH (300 µmol/L) in Hanks’ balanced salt solution (2 hours, 37°C) or to Ca²⁺ ionophore (10 µmol/L) in modified Hanks’ balanced salt solution containing 2.5 mmol/L CaCl₂ and 10 mmol/L HEPES (45 minutes, 37°C).^1,4 Induction of apoptosis was performed by inhibition of anchorage-dependent cell spreading with the use of 1% BSA.²⁵ MVs and apoptotic bleb–containing culture supernatants were cleared from detached cells and debris (centrifugation at 500g, 10 minutes), pelleted by ultracentrifugation (100 000g, 90 minutes, 4°C),^1,3 washed, and resuspended in PBS containing 0.01% BHT. Viability was examined by using trypan blue (0.4%). Apoptosis was confirmed by cell morphology, DNA fragmentation, or annexin V staining.^26,27 For oxidation in vitro, MVs derived from Ca²⁺ ionophore–treated ECs were treated with t-BuOOH (30 µmol/L) and Fe²⁺ (5 µmol/L) for 75 minutes at 37°C. BHT (0.01% final concentration) was added to all vesicle preparations. Protein content of MVs was determined by using Coomassie Reagent (Pierce). Lipid phosphorus content was measured as described.²⁸ Lipids isolated from 10 µg of MV protein contained 0.1 µg phosphorus. All preparations were examined for endotoxin content by Limulus amoebocyte lysate assay. Only preparations containing <50 pg/mL endotoxin were used. To further exclude the effects of endotoxin, in some experiments polymyxin B (50 µg/mL) was added.¹¹

Microscopy
Membrane vesiculation was observed by light microscopy with the use of a Leitz Fluovert microscope at x320 magnification. For electron microscopy, MV pellets were prefixed in 2.5% glutaraldehyde and 2% formaldehyde in sodium cacodylate buffer (0.1 mol/L) for 45 minutes, rinsed in sodium cacodylate buffer, postfixed in 0.75% osmium tetroxide for 30 minutes, rinsed in water, dehydrated with 2,2-dimethoxypropane (15 minutes), and embedded in Spurr’s epoxy resin. Sections were made with a diamond knife on a Reichert Ultracut, mounted on dioxane Formvar (Merck)–coated copper grids, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM 902.

Lipid Analysis
Total lipids were extracted according to Bligh and Dyer²⁹ and separated into neutral lipids, fatty acids, and polar lipids by thin-layer chromatography on silica gel 60W plates (Merck) with hexane/diethyl ether/acetic acid (70:30:1). Lipids were visualized by iodine vapor and extracted by using methanol/chloroform/water (50:25:20).

Electrospray Ionization–Mass Spectrometry
Electrospray ionization–mass spectrometry was performed with a VG Quattro mass spectrometer (Micromass) connected to a PU-980 high-performance liquid chromatographic pump (0.1 mL/min, Jasco), an LG-980-02 Ternary Unit (Jasco), and a 10-port injection valve (VICI AG). Calibration was performed as described.²² Cone voltage was operated at 65 V, and capillary voltage was operated at 4.5 kV in the positive mode. Phospholipids were dissolved in acetonitrile/water/formic acid (50:50:0.1) and introduced by flow injection (0.1 mL/min).²² For quantification, dimyristoylphosphatidylcholine was used as an internal standard.^30,31 Phospholipids were quantified on the basis of their ion intensity relative to the ion intensity of the internal standard.^30–32

Leukocyte Adhesion Assays
Adhesion of U-937 cells or HL-60 cells to ECs was performed as described previously.³³ U-937 and HL-60 used in the adhesion assays were shown to behave like human monocytes and neutrophils, respectively.^11,33 Briefly, confluent ECs were incubated in 48-well plates with MVs or lipids resuspended in medium 199 containing 10% supplemented calf serum for 4 hours at 37°C. After washing of ECs, unstimulated U-937 or HL-60 cells (10⁵ cells per well) were added to ECs for 15 minutes, and adherent cells were counted.

Cell ELISA
Confluent ECs in 96-well plates were treated for 4 hours at 37°C. The assay was then performed as described previously.³⁴

In all leukocyte adhesion assays and ELISA experiments, TNF- (10 U/mL) was used as a positive control and yielded a reproducible 3- to 4-fold increase. Control indicates cells treated with medium only.

Flow Cytometry
MVs (0.25 µg vesicle protein per microliter) were incubated with antibodies (25 µg/mL in PBS, 1% BSA) for 30 minutes at 37°C. Primary antibody binding was detected with a biotinylated goat anti-mouse-IgM (1:500 dilution) followed by a streptavidin–Alexa Fluor 488 conjugate (1:500, Molecular Probes) with use of a Becton Dickinson fluorescence-activated cell sorter. To control for unspecific binding, the primary antibody was omitted in control experiments.

Statistical Analysis
One-way ANOVA and single regression were performed. Values of P<0.05 were considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Oxidized MVs and Apoptotic Blebs Stimulate Monocyte-Endothelial Interactions
Four different types of MVs and apoptotic blebs were used for stimulation of ECs: (1) MVs derived by treatment with t-BuOOH (t-BuMVs), (2) MVs derived by treatment with Ca²⁺ ionophore (Ca²⁺-ionoMVs), (3) MVs derived by treatment with Ca²⁺ ionophore and then oxidized in vitro (in vitro oxMVs), and (4) blebs derived from apoptotic ECs (apoptotic blebs).

Five to 10 minutes after treatment with t-BuOOH (300 µmol/L), ECs started to release round-shaped MVs (Figure 1A). After 2 hours, 50% to 60% of ECs formed vesicles with a loss of viability of 10% (not shown). In accordance with previous reports,³⁵ no signs of apoptosis were detected (not shown).

View larger version (60K): [in this window] [in a new window]   Figure 1. A, Membrane vesiculation of ECs treated with t-BuOOH can be observed by light microscopy. B, Media obtained from ECs that had been treated with t-BuOOH were added to a second set of confluent ECs before and after ultracentrifugation and monocyte binding assays were performed. CO indicates control (cells treated with medium only). Data are expressed as percentage of control (mean±SEM). C, Electron microscopy of pellets showed large sealed MVs with intact lipid bilayer.

Media obtained from stimulated ECs induced monocyte adhesion (Figure 1B), whereas media from untreated ECs were inactive (data not shown). Ultracentrifugation resulted in a significant loss of biological activity, indicating that biologically active MV had been released after treatment with t-BuOOH (Figure 1B). After ultracentrifugation, vesicle pellets were examined by electron microscopy, demonstrating large (up to 2.5 µm in diameter), sealed, round-shaped MVs with an intact lipid bilayer (Figure 1C). Other structures seen may be vesicle fragments that were caused by rupture of MVs during workup.

Isolated t-BuMVs concentration-dependently induced monocyte adhesion (Figure 2A). We hypothesized that t-BuMVs were oxidized and therefore biologically active. To test this hypothesis, ECs were treated with Ca²⁺ ionophore, which causes vesiculation (similar to Figure 1A),⁵ presumably not involving oxidation. Viability was >92% after 2 hours, and ECs did not undergo apoptosis (data not shown). Ca²⁺-ionoMVs did not induce monocyte adhesion (Figure 2B). To prove the importance of oxidation of MVs to induce monocyte-EC interactions, Ca²⁺-ionoMVs were oxidized with t-BuOOH (30 µmol/L) and iron (5 µmol/L) in vitro. As hypothesized, in vitro oxMVs now stimulated monocyte binding (Figure 2C).

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of MVs on monocyte adhesion to ECs. Confluent ECs were incubated with t-BuMVs (A), Ca2+-ionoMVs (Ca2+-iono vesicles, B), or in vitro oxMVs (in vitro ox vesicles, C) for 4 hours. t-BuOOH and Fe2+ were added to ECs according to the amount contained in MVs that had been oxidized in vitro (C). Monocyte binding data (representative of 3 separate experiments) are expressed as percentage of control (mean±SEM). *P<0.03.

Polar Lipids Contained in Oxidized MVs Specifically Stimulate Monocyte-EC Interactions
Total lipid extracts from t-BuMVs and in vitro oxMVs, but not lipid extracts from Ca²⁺-ionoMVs, stimulated ECs to bind to monocytes (Figure 3A). Aqueous phases did not induce monocyte adhesion (data not shown). Only the phospholipid fraction of t-BuMVs and in vitro oxMVs induced monocyte adhesion to ECs, whereas fatty acids and neutral lipids were not active (Figure 3B). Neither fraction of Ca²⁺-ionoMVs was biologically active (Figure 3B).

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of lipids extracted from MVs. Confluent ECs were incubated with lipids derived from t-BuMVs, Ca2+-ionoMVs, or in vitro oxMVs. Total lipid extracts derived from indicated amounts of vesicle protein (A) and fatty acids (FA), neutral lipids (NL), and polar lipids (PL) isolated from lipid extracts (B) were added to ECs. Monocyte binding data (representative of 3 separate experiments) are expressed as percentage of control (mean±SEM). *P<0.01.

Moreover, although total lipid extracts from in vitro oxMVs, but not those from Ca²⁺-ionoMVs, induced monocyte adhesion to ECs, neither of the 2 groups of MVs induced neutrophil adhesion or E-selectin expression on ECs (Table).

View this table: [in this window] [in a new window]   Table 1. Oxidized MVs Specifically Induce Monocyte Adhesion to ECs

Determination of Active Component in Oxidized MVs and Apoptotic Blebs
Previous studies have shown that PAF receptor antagonists inhibited the action of Ox-PAPC on ECs.^36,37 In this study, monocyte adhesion induced by oxidized MVs was significantly reduced by 2 PAF receptor antagonists, BN-52021 and CV-3988 (not shown). According to previously published data,³⁶ monocyte adhesion induced by Ox-PAPC was also reduced by BN-52021 and CV-3988 (not shown).

Quantification of the recently identified biologically active oxidized phospholipids POVPC (mass-to-charge ratio [m/z] 594.3), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC, m/z 610.2), and 1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphocholine (PEIPC, m/z 828.5)^22,38 as well as lysophosphatidylcholine (lyso-PC, m/z 496) and PAPC (m/z 782) in oxidized and native MVs showed that POVPC was increased 5.4-fold and lyso-PC was increased 2-fold in MVs oxidized in vitro compared with native MVs. The increase in levels of PGPC or PEIPC in oxidized MVs was much less pronounced. Levels of PAPC were decreased, consistent with the increased formation of the oxidized compounds. Each analyzed MV preparation was also tested for monocyte binding. Using a single regression model, we found that levels of POVPC showed a fairly strong association (r²=0.463, P<0.0001) and that lyso-PC showed a weak positive correlation (r²=0.206, P<0.0103) with the extent of monocyte adhesion induced by MVs. Levels of PAPC (r²=0.027, P<0.3802), PGPC (r²=0.085, P<0.111), and PEIPC (r²=0.179, P<0.177) were not correlated with biological activity.

These data suggest that POVPC might play a dominant role in mediating monocyte binding. Thus, we examined whether IgM monoclonal antibodies that bind to POVPC (E03 and E06) would recognize MVs. We show that E03 and E06 bind to oxidized MVs (Figure 4A and 4B) and much less to native MVs (not shown). Furthermore, the IgM monoclonal E014, which recognizes malondialdehyde-lysine epitopes, also binds strongly to the oxidized MVs (Figure 4C).

View larger version (21K): [in this window] [in a new window]   Figure 4. Binding of E0 antibodies to MVs analyzed by flow cytometry. Binding of E03 (A), E06 (B), and E014 (C) to in vitro oxMVs (white area) was compared with binding of the secondary antibody only (black area). Data are representative of 2 separate experiments.

It has been shown that these antibodies bind to the surface of cells undergoing apoptosis and recognize apoptotic blebs,³⁹ indicating that apoptotic blebs also contain oxidized phospholipids. Indeed, apoptotic blebs derived from ECs induced monocyte binding (Figure 5A). The biological activity resided in the lipid fraction (Figure 5B).

View larger version (19K): [in this window] [in a new window]   Figure 5. Apoptotic blebs stimulate ECs to bind monocytes. Confluent ECs were incubated with indicated concentrations of isolated apoptotic blebs (A) or lipid extracts derived from the indicated amount of apoptotic blebs (B) for 4 hours. Monocyte binding data (representative of 3 separate experiments) are expressed as percentage of control (mean±SEM). *P<0.01.

Furthermore, we show that the ability of apoptotic blebs or MVs to induce monocyte adhesion was abolished by preincubation with E06 or E03 (Figure 6A and 6B). Similarly, E06 and E03 abolished the biological activity of lipid extracts of in vitro oxMVs (Figure 6C). In contrast, preincubation with E014, which was also strongly bound to oxidized MVs, had no effect, nor did an irrelevant IgM against tissue plasminogen activator (Figure 6). Furthermore, E03 and E06 blocked monocyte adhesion induced by POVPC, whereas E014 and the irrelevant antibody had no effect (Figure 6D). No inhibitory effects of any antibody on TNF-–induced monocyte adhesion to ECs were observed (data not shown). Finally, E03, E06, E014, and the irrelevant antibody alone had no effect on monocyte adhesion (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 6. Effect of E0 antibodies on stimulation of ECs. Apoptotic blebs (A), in vitro oxMVs (in vitro ox ves, B), lipid extracts of in vitro oxMVs (lipid extracts of in vitro ox ves, C), and POVPC (D) were preincubated in the presence or absence of monoclonal antibodies (E03, E06, E014, or an irrelevant IgM antibody [irr.]) for 30 minutes and then added to confluent ECs. Data from monocyte adhesion assays (mean of 3 separate experiments) are expressed as percentage of control (mean±SEM). *P<0.02.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
It has been shown that levels of MVs and apoptotic blebs derived from various cell types were increased in patients with vascular disease.^{3,14,15,17–20} Although a role for MVs in inflammatory processes has been implied, molecules responsible for the activation of vascular cells remain to be identified. In the present study, we demonstrate that oxidative modification of MVs led to the formation of lipid oxidation products that stimulated ECs to specifically bind monocytes, an important event in atherogenesis and other chronic inflammatory diseases. In contrast, MVs that were obtained by treatment of ECs with Ca²⁺ ionophore did not show biological activity. In addition, membrane blebs derived from ECs that underwent apoptosis also contained these lipid oxidation products and were biologically active.

It is important to note that the process of membrane vesiculation can be distinguished from membrane blebbing during apoptosis.^5,8,35 It has been shown that incubation of ECs with high doses of t-BuOOH (up to 0.4 mmol/L) results in membrane vesiculation and loss of cell viability but fails to induce apoptosis.³⁵ In the present study, the treatment of ECs with t-BuOOH resulted in no significant loss of viability for up to 2 hours, and no signs of apoptosis were detected. However, we cannot exclude the possibility that some of the isolated vesicles after t-BuOOH treatment are indeed blebs from apoptotic cells.

Oxidation of PAPC results in the formation of biologically active oxidized phospholipids, which are present in minimally modified (MM)-LDL^22,38,40 and atherosclerotic lesions²² and in mice fed a high-fat diet.^31,41 PAF receptor antagonists inhibited the activities of Ox-PAPC in vitro³⁶ and in vivo.³⁷ In the present study, characterization of the active component of oxidized MVs and apoptotic blebs revealed that polar lipids in these vesicles were responsible for their biological activity. Quantitative analysis of the polar lipid fraction by electrospray ionization–mass spectrometry showed increased levels of the oxidized phospholipids POVPC, PEIPC, and PGPC^22,38 in oxidized MVs compared with native MVs. Among these oxidized phospholipids, POVPC was shown to mimic the overall action of MM-LDL in terms of induction of leukocyte-endothelial interactions.^33,42 Like MM-LDL, oxidized MVs stimulated ECs to specifically bind monocytes but not neutrophils. Furthermore, E-selectin, the major adhesion molecule involved in neutrophil adhesion to ECs, was not expressed on ECs treated with oxidized MVs. Thus, these effects show striking similarity to the biological effects of MM-LDL and Ox-PAPC on ECs and imply a role for POVPC.

There are several indications that in the present study, oxovaleroyl-containing phospholipids play a predominant role. First, regression analysis of the levels of POVPC in MVs with the capability of inducing monocyte-EC interactions showed a highly significant correlation. Second, autoantibodies from apoE-deficient mice, which specifically recognize POVPC, block the action of oxidized MVs. Third, 2 different PAF receptor antagonists, which were shown to inhibit the action of POVPC,³⁷ also blocked monocyte adhesion induced by oxidized MVs.

The importance of oxidized phospholipids is further underlined by the fact that in apoE-deficient mice, which spontaneously develop atherosclerosis, high titers of autoantibodies to various oxidation-specific epitopes of oxidized LDL have been found.⁴³ Some of these autoantibodies recognize specific epitopes, such as malondialdehyde-lysine (E014) or POVPC (E03 and E06).⁴⁴ It has been demonstrated that both the POVPC-binding autoantibodies (E03 and E06) and E014 bind to apoptotic blebs³⁹ as well as to oxidized MVs. Despite this, only E03 and E06 blocked the stimulation of ECs to bind monocytes by oxidized MVs. These results provide strong evidence for POVPC and other oxovaleroyl-containing phospholipids contributing to biological activity and suggest that natural E0 autoantibodies might play a role in modulating the effect of oxidized MVs and apoptotic blebs in vivo. Conceivably, these antibodies might provide a valuable therapeutical tool by blocking the stimulation of ECs induced by oxidized MVs.

We propose that at sites of inflammation, where increased oxidant stress occurs, oxidized MVs are potentially released from various cell types. Furthermore, MVs that were shown to circulate at basal levels in the peripheral blood³ are potentially oxidized in the setting of systemic inflammation. Apoptotic blebs are generated at increased levels in various pathological conditions (see reviews^8,21). Thus, in addition to oxidized lipoproteins, which are believed to play a crucial role in the development of the atherosclerotic lesion, oxidized MVs and apoptotic blebs represent novel sources for biologically active oxidized phospholipids. Therefore, oxidized MVs and apoptotic blebs may contribute to the initiation and progression of chronic inflammatory processes and the development of atherosclerotic lesions.

	Acknowledgments

Top Abstract Introduction Methods Results Discussion References

 
This project was funded by the Austrian Science Foundation, project No. P13954-MED, and by the Interdisciplinary Cooperation-Project Program of the Austrian Federal Ministry for Education, Science, and Culture. We thank Dr Zyhdi Zhegu for preparation of human umbilical vein ECs and Reingard Ecker for excellent technical assistance.

Received July 2, 2001; accepted October 10, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Hamilton KK, Hattori R, Esmon CT, Sims PJ. Complement proteins C5b-9 induce vesiculation of the endothelial plasma membrane and expose catalytic surface for assembly of the prothrombinase enzyme complex. J Biol Chem. 1990; 265: 3809–3814.[Abstract/Free Full Text]

2. Fourcade O, Simon MF, Viode C, Rugani N, Leballe F, Ragab A, Fournie B, Sarda L, Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell. 1995; 80: 919–927.[CrossRef][Medline] [Order article via Infotrieve]

3. Combes V, Simon AC, Grau GE, Arnoux D, Camoin L, Sabatier F, Mutin M, Sanmarco M, Sampol J, Dignat GF. In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest. 1999; 104: 93–102.[Medline] [Order article via Infotrieve]

4. Patel KD, Zimmerman GA, Prescott SM, McIntyre TM. Novel leukocyte agonists are released by endothelial cells exposed to peroxide. J Biol Chem. 1992; 267: 15168–15175.[Abstract/Free Full Text]

5. Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood. 1997; 89: 1121–1132.[Free Full Text]

6. Miyoshi H, Umeshita K, Sakon M, Imajoh OS, Fujitani K, Gotoh M, Oiki E, Kambayashi J, Monden M. Calpain activation in plasma membrane bleb formation during tert-butyl hydroperoxide-induced rat hepatocyte injury [published erratum appears in Gastroenterology. 1996;111:1402]. Gastroenterology. 1996; 110: 1897–1904.[CrossRef][Medline] [Order article via Infotrieve]

7. Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol. 2001; 3: 339–345.[CrossRef][Medline] [Order article via Infotrieve]

8. Mallat Z, Tedgui A. Current perspective on the role of apoptosis in atherothrombotic disease. Circ Res. 2001; 88: 998–1003.[Abstract/Free Full Text]

9. Zamzani N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B, Kroemer G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med. 1995; 182: 367–377.[Abstract/Free Full Text]

10. Barry OP, Pratico D, Lawson JA, FitzGerald GA. Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles. J Clin Invest. 1997; 99: 2118–2127.[Medline] [Order article via Infotrieve]

11. Barry OP, Pratico D, Savani RC, FitzGerald GA. Modulation of monocyte-endothelial cell interactions by platelet microparticles. J Clin Invest. 1998; 102: 136–144.[Medline] [Order article via Infotrieve]

12. Mesri M, Altieri DC. Leukocyte microparticles stimulate endothelial cell cytokine release and tissue factor induction in a JNK1 signaling pathway. J Biol Chem. 1999; 274: 23111–23118.[Abstract/Free Full Text]

13. Forlow SB, McEver RP, Nollert MU. Leukocyte-leukocyte interactions mediated by platelet microparticles under flow. Blood. 2000; 95: 1317–1323.[Abstract/Free Full Text]

14. Mallat Z, Benamer H, Hugel B, Benessiano J, Steg PG, Freyssinet JM, Tedgui A. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation. 2000; 101: 841–843.[Abstract/Free Full Text]

15. Nomura S, Suzuki M, Katsura K, Xie GL, Miyazaki Y, Miyake T, Kido H, Kagawa H, Fukuhara S. Platelet-derived microparticles may influence the development of atherosclerosis in diabetes mellitus. Atherosclerosis. 1995; 116: 235–240.[CrossRef][Medline] [Order article via Infotrieve]

16. Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC, Maquelin KN, Roozendaal KJ, Jansen PG, ten Have K, Eijsman L, Hack CE, Sturk A. Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant. Circulation. 1997; 96: 3534–3541.[Abstract/Free Full Text]

17. Mallat Z, Hugel B, Ohan J, Leseche G, Freyssinet JM, Tedgui A. Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity. Circulation. 1999; 99: 348–353.[Abstract/Free Full Text]

18. Holme PA, Solum NO, Brosstad F, Roger M, Abdelnoor M. Demonstration of platelet-derived microvesicles in blood from patients with activated coagulation and fibrinolysis using a filtration technique and Western blotting. Thromb Haemost. 1994; 72: 666–671.[Medline] [Order article via Infotrieve]

19. Hugel B, Socie G, Vu T, Toti F, Gluckman E, Freyssinet JM, Scrobohaci ML. Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia. Blood. 1999; 93: 3451–3456.[Abstract/Free Full Text]

20. Warkentin TE, Hayward CP, Boshkov LK, Santos AV, Sheppard JA, Bode AP, Kelton JG. Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia. Blood. 1994; 84: 3691–3699.[Abstract/Free Full Text]

21. Freyssinet JM, Toti F, Hugel B, Gidon JC, Kunzelmann C, Martinez MC, Meyer D. Apoptosis in vascular disease. Thromb Haemost. 1999; 82: 727–735.[Medline] [Order article via Infotrieve]

22. Watson AD, Leitinger N, Navab M, Faull KF, Horkko S, Witztum JL, Palinski W, Schwenke D, Salomon RG, Sha W, et al. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo. J Biol Chem. 1997; 272: 13597–13607.[Abstract/Free Full Text]

23. Ravandi A, Kuksis A, Myher JJ, Marai L. Determination of lipid ester ozonides and core aldehydes by high-performance liquid chromatography with on-line mass spectrometry. J Biochem Biophys Methods. 1995; 30: 271–285.[CrossRef][Medline] [Order article via Infotrieve]

24. Zhang JC, Fabry A, Paucz L, Wojta J, Binder BR. Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells. Blood. 1996; 88: 3880–3886.[Abstract/Free Full Text]

25. Re F, Zanetti A, Sironi M, Polentarutti N, Lanfrancone L, Dejana E, Colotta F. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol. 1994; 127: 537–546.[Abstract/Free Full Text]

26. Stehlik C, de Martin R, Kumabashiri I, Schmid JA, Binder BR, Lipp J. Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis. J Exp Med. 1998; 188: 211–216.[Abstract/Free Full Text]

27. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995; 182: 1545–1556.[Abstract/Free Full Text]

28. Zhou X, Arthur G. Improved procedures for the determination of lipid phosphorus by malachite green. J Lipid Res. 1992; 33: 1233–1236.[Abstract]

29. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Physiol. 1959; 37: 911–917.

30. Han X, Gross RW. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids. Proc Natl Acad Sci U S A. 1994; 91: 10635–10639.[Abstract/Free Full Text]

31. Leitinger N, Watson AD, Hama SY, Ivandic B, Qiao JH, Huber J, Faull KF, Grass DS, Navab M, Fogelman AM, et al. Role of group II secretory phospholipase A₂ in atherosclerosis, 2: potential involvement of biologically active oxidized phospholipids. Arterioscler Thromb Vasc Biol. 1999; 19: 1291–1298.[Abstract/Free Full Text]

32. Han X, Gubitosi KR, Collins BJ, Gross RW. Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis. Biochemistry. 1996; 35: 5822–5832.[CrossRef][Medline] [Order article via Infotrieve]

33. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson M, Fogelman AM. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest. 1990; 85: 1260–1266.[Medline] [Order article via Infotrieve]

34. Krebs M, Kaun C, Lorenz M, Haag WM, Geiger M, Binder BR. Protease dependent activation of endothelial cells by peritoneal dialysis effluents. Thromb Haemost. 1999; 82: 1334–1341.[Medline] [Order article via Infotrieve]

35. van Gorp RM, Broers JL, Reutelingsperger CP, Bronnenberg NM, Hornstra G, van-Dam-Mieras MC, Heemskerk JW. Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis. Am J Physiol. 1999; 277: C20–C28.[Medline] [Order article via Infotrieve]

36. Leitinger N, Watson AD, Faull KF, Fogelman AM, Berliner JA. Monocyte binding to endothelial cells induced by oxidized phospholipids present in minimally oxidized low density lipoprotein is inhibited by a platelet activating factor receptor antagonist. Adv Exp Med Biol. 1997; 433: 379–382.[Medline] [Order article via Infotrieve]

37. Subbanagounder G, Leitinger N, Shih PT, Faull KF, Berliner JA. Evidence that phospholipid oxidation products and/or platelet-activating factor play an important role in early atherogenesis: in vitro and in vivo inhibition by WEB 2086. Circ Res. 1999; 85: 311–318.[Abstract/Free Full Text]

38. Watson AD, Subbanagounder G, Welsbie DS, Faull KF, Navab M, Jung ME, Fogelman AM, Berliner JA. Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein. J Biol Chem. 1999; 274: 24787–24798.[Abstract/Free Full Text]

39. Chang MK, Bergmark C, Laurila A, Horkko S, Han KH, Friedman P, Dennis EA, Witztum JL. Monoclonal antibodies against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: evidence that oxidation-specific epitopes mediate macrophage recognition. Proc Natl Acad Sci U S A. 1999; 96: 6353–6358.[Abstract/Free Full Text]

40. Watson AD, Berliner JA, Hama SY, La DB, Faull KF, Fogelman AM, Navab M. Protective effect of high density lipoprotein associated paraoxonase: inhibition of the biological activity of minimally oxidized low density lipoprotein. J Clin Invest. 1995; 96: 2882–2891.[Medline] [Order article via Infotrieve]

41. Berliner J, Leitinger N, Watson A, Huber J, Fogelman A, Navab M. Oxidized lipids in atherogenesis: formation, destruction and action. Thromb Haemost. 1997; 78: 195–199.[Medline] [Order article via Infotrieve]

42. Leitinger N, Tyner TR, Oslund L, Rizza C, Subbanagounder G, Lee H, Shih PT, Mackman N, Tigyi G, Territo MC, et al. Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils. Proc Natl Acad Sci U S A. 1999; 96: 12010–12015.[Abstract/Free Full Text]

43. Palinski W, Horkko S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice: demonstration of epitopes of oxidized low density lipoprotein in human plasma. J Clin Invest. 1996; 98: 800–814.[Medline] [Order article via Infotrieve]

44. Horkko S, Bird DA, Miller E, Itabe H, Leitinger N, Subbanagounder G, Berliner JA, Friedman P, Dennis EA, Curtiss LK, et al. Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid-protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins. J Clin Invest. 1999; 103: 117–128.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				K. Hartvigsen, M.-Y. Chou, L. F. Hansen, P. X. Shaw, S. Tsimikas, C. J. Binder, and J. L. Witztum The role of innate immunity in atherogenesis J. Lipid Res., April 1, 2009; 50(Supplement): S388 - S393. [Abstract] [Full Text] [PDF]

				R. Chen, X. Chen, R. G. Salomon, and T. M. McIntyre Platelet Activation by Low Concentrations of Intact Oxidized LDL Particles Involves the PAF Receptor Arterioscler Thromb Vasc Biol, March 1, 2009; 29(3): 363 - 371. [Abstract] [Full Text] [PDF]

				M. C. Plotkowski, L. F. P. Feliciano, G. B. S. Machado, L. G. Cunha Jr, C. Freitas, A. M. Saliba, and M. C. de Assis ExoU-induced procoagulant activity in Pseudomonas aeruginosa-infected airway cells Eur. Respir. J., December 1, 2008; 32(6): 1591 - 1598. [Abstract] [Full Text] [PDF]

				C. Bergmark, A. Dewan, A. Orsoni, E. Merki, E. R. Miller, M.-J. Shin, C. J. Binder, S. Horkko, R. M. Krauss, M. J. Chapman, et al. A novel function of lipoprotein [a] as a preferential carrier of oxidized phospholipids in human plasma J. Lipid Res., October 1, 2008; 49(10): 2230 - 2239. [Abstract] [Full Text] [PDF]

				C. Peter, M. Waibel, C. G. Radu, L. V. Yang, O. N. Witte, K. Schulze-Osthoff, S. Wesselborg, and K. Lauber Migration to Apoptotic "Find-me" Signals Is Mediated via the Phagocyte Receptor G2A J. Biol. Chem., February 29, 2008; 283(9): 5296 - 5305. [Abstract] [Full Text] [PDF]

				E. Biro, R. Nieuwland, P. P Tak, L. M Pronk, M. C L Schaap, A. Sturk, and C E. Hack Activated complement components and complement activator molecules on the surface of cell-derived microparticles in patients with rheumatoid arthritis and healthy individuals Ann Rheum Dis, August 1, 2007; 66(8): 1085 - 1092. [Abstract] [Full Text] [PDF]

				F. Gruber, O. Oskolkova, A. Leitner, M. Mildner, V. Mlitz, B. Lengauer, A. Kadl, P. Mrass, G. Kronke, B. R. Binder, et al. Photooxidation Generates Biologically Active Phospholipids That Induce Heme Oxygenase-1 in Skin Cells J. Biol. Chem., June 8, 2007; 282(23): 16934 - 16941. [Abstract] [Full Text] [PDF]

				S. J. Germain, G. P. Sacks, S. R. Soorana, I. L. Sargent, and C. W. Redman Systemic Inflammatory Priming in Normal Pregnancy and Preeclampsia: The Role of Circulating Syncytiotrophoblast Microparticles J. Immunol., May 1, 2007; 178(9): 5949 - 5956. [Abstract] [Full Text] [PDF]

				X. Feng, H. Li, A. A. Rumbin, X. Wang, A. La Cava, K. Brechtelsbauer, L. W. Castellani, J. L. Witztum, A. J. Lusis, and B. P. Tsao ApoE-/-Fas-/- C57BL/6 mice: a novel murine model simultaneously exhibits lupus nephritis, atherosclerosis, and osteopenia J. Lipid Res., April 1, 2007; 48(4): 794 - 805. [Abstract] [Full Text] [PDF]

				A. A. Birukova, P. Fu, S. Chatchavalvanich, D. Burdette, O. Oskolkova, V. N. Bochkov, and K. G. Birukov Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L924 - L935. [Abstract] [Full Text] [PDF]

				O. Morel, F. Toti, B. Hugel, B. Bakouboula, L. Camoin-Jau, F. Dignat-George, and J.-M. Freyssinet Procoagulant Microparticles: Disrupting the Vascular Homeostasis Equation? Arterioscler Thromb Vasc Biol, December 1, 2006; 26(12): 2594 - 2604. [Abstract] [Full Text] [PDF]

				C. M. Boulanger, N. Amabile, and A. Tedgui Circulating Microparticles: A Potential Prognostic Marker for Atherosclerotic Vascular Disease Hypertension, August 1, 2006; 48(2): 180 - 186. [Full Text] [PDF]

				J. Huber, A. Furnkranz, V. N. Bochkov, M. K. Patricia, H. Lee, C. C. Hedrick, J. A. Berliner, B. R. Binder, and N. Leitinger Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase J. Lipid Res., May 1, 2006; 47(5): 1054 - 1062. [Abstract] [Full Text] [PDF]

				S. Bluml, S. Kirchberger, V. N. Bochkov, G. Kronke, K. Stuhlmeier, O. Majdic, G. J. Zlabinger, W. Knapp, B. R. Binder, J. Stockl, et al. Oxidized Phospholipids Negatively Regulate Dendritic Cell Maturation Induced by TLRs and CD40 J. Immunol., July 1, 2005; 175(1): 501 - 508. [Abstract] [Full Text] [PDF]

				B. Hugel, M. C. Martinez, C. Kunzelmann, and J.-M. Freyssinet Membrane Microparticles: Two Sides of the Coin Physiology, February 1, 2005; 20(1): 22 - 27. [Abstract] [Full Text] [PDF]

				H. S. Goodridge, F. A. Marshall, K. J. Else, K. M. Houston, C. Egan, L. Al-Riyami, F.-Y. Liew, W. Harnett, and M. M. Harnett Immunomodulation via Novel Use of TLR4 by the Filarial Nematode Phosphorylcholine-Containing Secreted Product, ES-62 J. Immunol., January 1, 2005; 174(1): 284 - 293. [Abstract] [Full Text] [PDF]

				M.-K. Chang, C. J. Binder, Y. I. Miller, G. Subbanagounder, G. J. Silverman, J. A. Berliner, and J. L. Witztum Apoptotic Cells with Oxidation-specific Epitopes Are Immunogenic and Proinflammatory J. Exp. Med., December 6, 2004; 200(11): 1359 - 1370. [Abstract] [Full Text] [PDF]

				K. G. Birukov, V. N. Bochkov, A. A. Birukova, K. Kawkitinarong, A. Rios, A. Leitner, A. D. Verin, G. M. Bokoch, N. Leitinger, and Joe. G.N. Garcia Epoxycyclopentenone-Containing Oxidized Phospholipids Restore Endothelial Barrier Function via Cdc42 and Rac Circ. Res., October 29, 2004; 95(9): 892 - 901. [Abstract] [Full Text] [PDF]

				M. Yeh, A. L. Cole, J. Choi, Y. Liu, D. Tulchinsky, J.-H. Qiao, M. C. Fishbein, A. N. Dooley, T. Hovnanian, K. Mouilleseaux, et al. Role for Sterol Regulatory Element-Binding Protein in Activation of Endothelial Cells by Phospholipid Oxidation Products Circ. Res., October 15, 2004; 95(8): 780 - 788. [Abstract] [Full Text] [PDF]

				M. Navab, G. M. Ananthramaiah, S. T. Reddy, B. J. Van Lenten, B. J. Ansell, G. C. Fonarow, K. Vahabzadeh, S. Hama, G. Hough, N. Kamranpour, et al. Thematic review series: The Pathogenesis of Atherosclerosis The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL J. Lipid Res., June 1, 2004; 45(6): 993 - 1007. [Abstract] [Full Text] [PDF]

				J. Watanabe, G. K. Marathe, P. O. Neilsen, A. S. Weyrich, K. A. Harrison, R. C. Murphy, G. A. Zimmerman, and T. M. McIntyre Endotoxins Stimulate Neutrophil Adhesion Followed by Synthesis and Release of Platelet-activating Factor in Microparticles J. Biol. Chem., August 29, 2003; 278(35): 33161 - 33168. [Abstract] [Full Text] [PDF]

				K. A. Walton, X. Hsieh, N. Gharavi, S. Wang, G. Wang, M. Yeh, A. L. Cole, and J. A. Berliner Receptors Involved in the Oxidized 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine-mediated Synthesis of Interleukin-8: A ROLE FOR TOLL-LIKE RECEPTOR 4 AND A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN J. Biol. Chem., August 8, 2003; 278(32): 29661 - 29666. [Abstract] [Full Text] [PDF]

				M. J. VanWijk, E. VanBavel, A. Sturk, and R. Nieuwland Microparticles in cardiovascular diseases Cardiovasc Res, August 1, 2003; 59(2): 277 - 287. [Abstract] [Full Text] [PDF]

				N. Mackman How Do Oxidized Phospholipids Inhibit LPS Signaling? Arterioscler Thromb Vasc Biol, July 1, 2003; 23(7): 1133 - 1136. [Full Text] [PDF]

				K. A. Walton, A. L. Cole, M. Yeh, G. Subbanagounder, S. R. Krutzik, R. L. Modlin, R. M. Lucas, J. Nakai, E. J. Smart, D. K. Vora, et al. Specific Phospholipid Oxidation Products Inhibit Ligand Activation of Toll-Like Receptors 4 and 2 Arterioscler Thromb Vasc Biol, July 1, 2003; 23(7): 1197 - 1203. [Abstract] [Full Text] [PDF]

				P. X. Shaw, C. S. Goodyear, M.-K. Chang, J. L. Witztum, and G. J. Silverman34 The Autoreactivity of Anti-Phosphorylcholine Antibodies for Atherosclerosis-Associated Neo-Antigens and Apoptotic Cells J. Immunol., June 15, 2003; 170(12): 6151 - 6157. [Abstract] [Full Text] [PDF]

				H. Ishii, T. Tezuka, H. Ishikawa, K. Takada, K. Oida, and S. Horie Oxidized phospholipids in oxidized low-density lipoprotein down-regulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RAR{beta}-RXR{alpha} heterodimers and Sp1 and Sp3 to their binding sequences in the TM promoter Blood, June 15, 2003; 101(12): 4765 - 4774. [Abstract] [Full Text] [PDF]

				S. L. Hazen and G. M. Chisolm Oxidized phosphatidylcholines: Pattern recognition ligands for multiple pathways of the innate immune response PNAS, October 1, 2002; 99(20): 12515 - 12517. [Full Text] [PDF]

				M.-K. Chang, C. J. Binder, M. Torzewski, and J. L. Witztum From the Cover: C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand: Phosphorylcholine of oxidized phospholipids PNAS, October 1, 2002; 99(20): 13043 - 13048. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huber, J. Articles by Leitinger, N. Search for Related Content PubMed PubMed Citation Articles by Huber, J. Articles by Leitinger, N. Related Collections Apoptosis Pathophysiology Risk Factors Lipid and lipoprotein metabolism Mechanism of atherosclerosis/growth factors