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Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5
Accepted Manuscript Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5. Min Wan , Xiang Hua , Jun Su , Divya Thiagarajan , Anna G. Frostegård , Jesper Z. Haeggström , Johan Frostegård PII:
S0021-9150(14)01163-0
DOI:
10.1016/j.atherosclerosis.2014.05.913
Reference:
ATH 13538
To appear in:
Atherosclerosis
Received Date: 4 September 2013 Revised Date:
25 April 2014
Accepted Date: 1 May 2014
Please cite this article as: Wan M, Hua X, Su J, Thiagarajan D, Frostegård AG, Haeggström JZ, Frostegård J, Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5., Atherosclerosis (2014), doi: 10.1016/j.atherosclerosis.2014.05.913. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5. Min Wan1*, Xiang Hua2*#, Jun Su2, Divya Thiagarajan2, Anna G Frostegård2, Jesper Z. Haeggström1 and Johan Frostegård2
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Division of Physiological Chemistry II, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm, Sweden
IMM, Karolinska University Hospital, Huddinge, Karolinska Institutet, Stockholm, Sweden
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corresponding author
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#
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* contributed equally
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Objective
Cardiolipin (CL) is a phospholipid with an unusual dimeric structure containing four double-bonds and is easily oxidized. CL is present in mitochondria. Here we explored potential pro-inflammatory properties implicated in cardiovascular disease (CVD): activation of endothelial cells, 5lipoxygenase (5-LOX) and leukotriene B4 (LTB4), by oxidized CL (oxCL) and inhibitory effects of
Methods In
monocytes/macrophages
and
neutrophils,
calcium
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Annexin A5, an antithrombotic and antiinflammatory plasma protein.
mobilization
was
monitored
spectrophotometrically with Fura-2 and synthesis of LTB4 was analyzed by EIA. Expression of adhesion molecules on endothelial cells was studied by FACScan. Binding of Annexin A5 were
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analysed by ELISA. The mRNA expression of 5-LOX and cyclooxygenase-2 was assessed by RealTime PCR.
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Results
We demonstrate that oxCL but not its non-oxidized counterpart CL induces biosynthesis of LTB4 and increases intracellular concentrations of calcium in monocytes/macrophages and neutrophils. oxCL rather than CL selectively elevates gene expression of 5-LOX but not COX-2 in human macrophages. Furthermore, oxCL but not CL raises levels of adhesion molecules ICAM-1 and VCAM-1 in endothelial cells. Annexin A5 can bind oxCL to abolish all these oxCL-induced effects.
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Conclusions
oxCL may promote inflammation and related diseases especially in conditions involving unresolved apoptosis and necrosis, such as atherosclerosis, where free oxCL is likely to be released from liberated mitochondria. Increased intracellular calcium could activate 5-LOX to produce
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Leukotriene B4 (LTB4). Annexin A5 inhibits the pro-inflammatory effects of oxCL and its
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potential therapeutic use when oxCL is implicated in inflammation could be of interest.
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Introduction
Cardiolipin (CL) is a dimeric phospholipid which is known to be present in eucaryotic cells, bacteria and archaebacteria, however its functional role is only partly known1. It is more prevalent in cells with high metabolic activity, like heart and skeletal muscle, especially in mitochondrial membranes. The presence of CL in mitochondria and bacteria is interesting from an evolutionary point of view since mitochondria are likely to have a bacterial origin2.
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CL has a unique dimeric structure, highly enriched in linoleic acid moieties with double bonds, and susceptible to oxidation3, 4. It has been suggested that CL plays a role in generation of an electrochemical potential for substrate transport and ATP synthesis both in bacteria and mitochondria5, 6. CL that has undergone oxidation (oxCL) promotes delocalization and release of
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cytochrome c, predisposing to its release from mitochondria and the activation of cell death programmes4, 7, 8. Antibodies against CL (aCL) cause both venous and arterial thrombosis, and are known to be of major importance in rheumatic diseases, especially systemic lupus erythematosus
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(SLE), cardiovascular disease and venous thrombosis9.
Atherosclerosis, the major cause of CVD, is an inflammatory conditions, where macrophage- and endothelial cell activation are generally believed to play an important role. Also neutrophils have been discussed during recent years10. 5-LipoXygenase (5-LOX) has a calcium-binding domain, and increased intracellular calcium could activate 5-LOX to produce Leukotriene B4 (LTB4),
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which may play an important role in plaque rupture and other inflammation-related conditions11. Another important aspect of inflammation, in relation to atherosclerosis, is adhesion molecule expression on endothelial cells.
Annexin A5 is a member of the Annexin superfamily, and has anti-thrombotic properties due to
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interaction with phospholipids, especially phosphatidylserine, and thus inhibits the coagulation cascade. We recently demonstrated that antiCL suppressed binding of Annexin A5 to endothelial
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cells and suggested that Annexin A5 could have anti-atherothrombotic properties in SLE and in general12. In line with this Annexin A5 also has antiatherosclerotic properties, and modulates endothelial functions13, one mechanism could be inhibition of the major inflammatory phospholipid lysophosphatidylcholine14. We recently reported that enhanced levels of antibodies against oxCL (anti-OxCL), but not CL, are associated with an increased risk to develop CVD among 60-year olds in a population based study15 and that mortality among hemodialysis-patients where the risk of death from CVD is very high, is negatively associated with anti-OxCL16. Further, in SLE, where the risk of CVD is very high and the prevalence of atherosclerotic plaque is increased, we demonstrated a negative association between anti-OxCL and atherosclerotic plaques, including echolucent ones17. Our findings thus clearly indicate that oxCL as an antigen is present in humans, and also is
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generating immune responses among immune competent cells as B-cells. In principle oxCL could thus exert effects both in the atherosclerotic plaques and in the circulation or other tissues. OxCL is present in atherosclerotic lesions and apoptotic cells18 and is implicated in cell death4, 7, 8
, which is a major characteristic of atherosclerotic plaques. However, little is known about its
properties relevant to atherosclerosis and inflammation, which is the topic of the present study. We here report that oxCL in contrast to CL has pro-inflammatory properties, which could be inhibited
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atherosclerosis and its complications are discussed.
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by Annexin A5. The potential implications of these findings for inflammatory conditions including
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Materials and methods
Oxidation of CL CL from bovine heart was purchased as ethanol solution from Sigma (Sigma product C 1649) and was stored at –20° C. Hydro heart CL (reduced CL) was purchased from Avanti Polar Lipids, Inc. To generate saturated molecular species, CL was oxidized in aqueous solutions containing 1.5
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mmol/L tert-butylhydroperoxide and CuSO4 in 20 µmol/L, essentially as described. We have previously reported that CL treated according to this method was oxidatively modifed15.
Cell culture
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Human polymorphonuclear leukocytes (PMN)
Human PMNs were isolated from freshly prepared buffy coats (Karolinska Hospital blood bank,
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Stockholm, Sweden) by dextran sedimentation, hypotonic lysis of erythrocytes and gradient centrifugation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). PMNs were suspended at a density of 10 × 106/ml in Dulbecco’s PBS (Gibco (Invitrogen), Paisley, UK). PMN purity (> 95%) and viability (> 98%) was determined using Hemacolor (J.T. Baker, Utrecht, Holland) and Trypan Blue (Sigma Chemical Co.) staining, respectively.
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Human monocyte-derived macrophages (HMDM)
Human mononuclear cells were isolated from freshly prepared buffy coats (Karolinska Hospital blood bank, Stockholm, Sweden) by gradient centrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden). Differentiation of human monocytes to macrophages was achieved
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by plating mononuclear cells in cell culture plates for two hours, and then unbound cells were washed away with PBS, followed by cell culture over seven days in RPMI-1640 medium with
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25mM Hepes, 1% L-glutamine, 1% penicillin-streptomycin and 10% FBS.
Endothelial cells
Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2 were purchased from Cascade Biologics, Inc (Portland, Ore). Cultures were maintained in EGMTM phenol red-free medium (Clonetics, San Diego, Calif), containing 2% of fetal bovine serum and supplements, at 37°C under humidified 5% CO2 conditions. All experiments were performed at passage 3 to 5. HUVECs were seeded at density of 6 × 104 cells/2 mL on 6-well plates (NUNC Inc, Naperville, Ill). After HUVECs attachment overnight, cells were ready for stimulation.
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Intracellular calcium mobilization
Human mononuclear leukocytes and PMNs were isolated from buffy coat as described above. Human monocytes were cultured and differentiated into human macrophages in black, 96-well plates with transparent bottom (Corning Costar; 5 × 105 cells/well) for seven days, and PMNs were seeded into black, 96-well plates with transparent bottom (Corning Costar; 5 × 104 cells/well), and spin down the plate at 120 × g for 5 min. When human macrophages and neutrophils were ready to
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use, the medium containing 4 µM FURA-2AM (Fura-2 acetoxymethyl ester), or buffer as appropriate was added into each well, and incubated for 30 min at 37°C and 5% CO2. Thereafter, cells were washed four times with 50 µl of a buffer solution (135 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 11 mM glucose, 11 mM Hepes, pH 7.4) before a final 50 µl volume of
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buffer was added to each well. Afterwards,the plates were transferred to a fluorometer (Fluostar™, BMG Technologies), and 50 µl of different agonists according to experimental designs or buffer
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solution as control was injected into individual wells and the fluorescence values in each well were monitored for the next 120 seconds. Control wells containing cells that had not been exposed to FURA-2AM were used to subtract background auto-fluorescence. The results are given as the ratio of mean fluorescence intensity (MFI) between 340 and 380 nm, and normalized by control.
Analysis of leukotriene B4 biosynthesis by EIA kit
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HMDMs (1 × 106) or PMNs (1 × 106) were incubated with different agents according to the experimental design, and then quenched with an equal volume methanol. After acidification to pH 3-4, the samples were purified by solid-phase extraction (SupelcleanTM LC-18, Supelco) and eluted in methanol. After evaporation of solvent under a stream of nitrogen, the samples were re-
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suspended in EIA buffer. The level of LTB4 was determined with LTB4 EIA kit (Cayman
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Chemical) by using dilutions within the linear portion of the standard curve.
Detection of adhesion molecules After the cells were washed with complete medium, oxCL and reduced CL (10 µg/ml) were added. For annexin A5 inhibition study, oxCL was incubated with Annexin A5 (5 µg/ml; Bender MedSystems GmbH, Austria) for 30 min before addition to cells. After 24 h incubation, detached floating cells were carefully collected, and remaining attached cells were gently harvested into Falcon FACS tubes after incubating with 400µl of FACSmax (Genlantis, USA) buffer for 10 min at room temperature. After centrifuging at 1200 rpm for 5 min, cells were resuspended in 50 µl Cell Staining Buffer (Biolegend, USA), incubated with FcR block (Miltenyi Biotec, Germany) for 10 min and then stained with PE-conjugated anti-CD54 (eBioscience) and FITC-conjugated antiHuman CD106 (Becton, Dickinson) for 30 min on ice. After washing with PBS, the fluorescence 6
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intensity of cells was measured by LSR Fortessa equipped with FACS Diva version 6 (BD Biosciences, San Jose, CA, USA).
Enzyme-Linked Immunosorbent Assay (ELISA) for Annexin A5 binding to antigen F96 microtiter polysorp plates (Nunc, Roskilde Denmark), were coated with oxCL, CL or Hydro Heart cardiolipin (R- CL) (Biosearch Technologies, Inc, Ca, USA) by incubation at a concentration
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of 5 µg/ml overnight at 40C. After five washings with PBS, the plates were blocked with 2% PBSBSA for 2 h at room temperature. Annexin A5 were added and incubated for another one hour. After washing, bound annexin A5 was detected by incubating subsequently with rabbit anti-human annexin V polyclonal antibodies (1:2000, Hyphen Biomed, Andresy, France) and polyclonal goat
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anti-rabbit Immunoglobulins/AP (1:3000, DakoCytomation). The reaction was developed with alkaline phosphatase substrate (Sigma), and optical density (OD) was read at 405 nm with an
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ELISA Multiscan Plus spectrophotometer (Molecular Devices Emax, San Francisco).
RNA extraction and quantitative real-Time PCR
Extraction of total RNA was performed by using the RNA Mini kit (Bioline, London, UK) including an on-column DNase digestion step. RNA concentration and quality were assessed by Nanodrop (Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized from total RNA by
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using SuperScript II reverse transcriptase (Life Technologies, Paisley, UK), and real-time PCR was performed in a TaqMan 7300 instrument (Life Technologies). Normalizations were made to β-actin. The primer/probe pairs were obtained by Assay-on-Demand (Life Technologies, Paisley, UK):
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respectively.
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human 5-LO, and β-actin with assay IDs Hs00386528_ml, Hs00153133_m1, and Hs99999903_ml,
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Results:
Intracellular calcium mobilization The influence of oxCL on intracellular calcium mobilization of HMDMs and PMNs were studied. Compared to control solution and CL, oxCL induces significantly increased intracellular calcium level in HMDM and PMNs (Fig. 1A and B). However, oxCL-induced intracellular calcium
change of intracellular calcium (Fig. 1A and B). LTB4 production
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mobilization is totally inhibited by Annexin A5, while Annexin A5 alone does not induce any
HMDMs and PMNs challenged with oxCL generate LTB4 (Fig. 2A, B) in a concentrationdependent manner. Native CL does not exhibit similar responses. LTB4 production induced by
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oxCL is completely blocked by Annexin A5 (Fig. 2A and B). 5-LOX gene expression in macrophages is upregulated by oxCL
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In addition to induction of a prompt LTB4 release from human neutrophils and macrophages, oxCL treatment for 6 h enhances the expression of 5-LOX mRNA, while CL exhibits no significant effect. However, oxCL treatment does not alter the level of COX-2 gene expression (Fig 3).
Effects of oxCL on endothelial cells
To study whether oxCL can promote the expression of adhesion molecules in endothelial cells,
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HUVECs were incubated for 24 h with oxCL or native CL. Our results indicate that oxCL significantly elevates both ICAM-1 and VCAM-1 expression, while native CL does not show the same effects (Fig.4A). Both the increased expression of ICAM-1 and VCAM-1 induced by oxCL
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were significantly inhibited by Annexin A5 (Fig. 4B).
Binding of Annexin A5 to cardiolipin
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To test whether Annexin A5 can bind various forms of cardiolipin, oxCL, CL and R-CL (5ug/ml) were coated on ELISA plates overnight before different concentrations of Annexin A5 were added to plates. Results show that binding of Annexin A5 in to oxCL is significantly stronger than binding of Annexin A5 to overnight air exposed CL. Moreover, no binding between reduced CL (R-CL) and Annexin A5 was detected (Fig. 5).
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Discussion
We report that oxCL but not CL has proinflammatory properties. OxCL could trigger LTB4 production in both human monocyte-derived macrophages (HMDM) and neutrophils. Further evidence of oxCL-mediated cell activation came from experiments demonstrating intracellular Ca2+ mobilization in these cell types. Leukotrienes are paracrine lipid mediators that have potent proinflammatory activities. Thus, LTB4 is one of the most potent chemotactic agents known to date,
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particularly for inflammatory cells and also evokes adhesion and transmigration of leukocytes across the endothelial barrier. LTB4 is biosynthesized from arachidonic acid by the sequential action of (5-LOX/FLAP) and LTA4 hydrolase, mainly in cells of myeloid lineage, such as neutrophils and macrophage19. Leukotrienes are known to exert broad pro-inflammatory effects as a
actions of LTB4
20, 21
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part of the innate immune response and evidence is accumulating regarding the antimicrobial . More recently, however, the LTB4 signaling pathway was found to be
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important for linking early immune responses and the multiple classes of effector cells associated with acquired immunity22. Two G-protein coupled LTB4 receptors have been identified, BLT1 and BLT2, with high and low affinity for LTB4, respectively23, 24.
Our finding that oxCL can elicit LTB4 synthesis in macrophages is particularly interesting in the context of recent data implying LTB4 in atherosclerosis and CVD. Thus, a growing body of biochemical, histochemical and genetic data indicate that LTB4 plays an important role in the
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pathogenesis of human atherosclerosis and its sequelae, myocardial infarction and stroke25-28. Accordingly, mRNA levels for the three key proteins in LTB4 synthesis are significantly increased in human atherosclerotic plaque, and more pronounced in patients with ongoing symptoms of
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plaque instability29. Even though neutrophils are not commonly found in atherosclerotic lesions, recent findings indicate that they may still play a role, and are also detected in lesions using new techniques30. Interestingly, neutrophils accumulate at sites where plaque rupture are detected,
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suggesting a role in this major complication of atherosclerosis31. Interestingly, oxCL not only induces intracellular calcium mobilization resulting in a rapid LTB4 production, but also triggers further 5-LOX gene expression in human macrophages. In addition to 5-LOX, AA can be alternatively metabolized by cyclooxygenase (COX) coupled with metabolism by downstream enzymes into prostanoids32. The link of prostanoids and CVD has been widely documented33, 34. However, oxCL displays no effects on COX-2 gene expression in macrophages, which indicates that oxCL might selectively regulate the 5-LOX/leukotriene pathway. OxCL but not CL induced endothelial cells to express ICAM-1 and VCAM-1. Adhesion molecules play an important role to recruit monocytes into the intima in the artery, which is likely to be an early step in the inflammatory process that is characteristic of atherogenesis 10, 35. 9
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Also other evidence implies that oxCL could play a role in atherosclerosis and its complications. Even though the antigen recognized by a recently described monoclonal is not the same type of oxCL as used herein, it is still interesting to note that the apparently mildly oxidized form of CL was abundant in lesions18. In the oxidative environment present in lesions, it is possible that further oxidation of CL occurs, even though this is not studied here. Another recent observation is that oxCL competes with oxLDL for uptake into macrophages
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through scavenger receptors15. Such macrophages are believed to transform into foam cells, which are relatively inert, and constitute much of the cell debris in the atherosclerotic lesions and appear to promote atherogenesis10,
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. Another feature of atherosclerosis is apoptosis and necrosis, where
typically, apoptotic cells are not cleared properly, but instead appear to become necrotic. Dying
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cells activate the innate immune system and promote an inflammatory response with release of the proinflammatory cytokine IL-1ß, and the inflammasome37. According to the danger hypothesis,
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endogenous factors released during cell death such as high-mobility group protein B1 (HMGB-1), double-stranded DNA, amyloid-β-peptide and heat-shock proteins (HSP), could promote inflammation38. If mitochondria, leaking from abundant dead cells in atherosclerotic lesions or in other locations, liberate oxCL, this could be yet another pro-inflammatory mechanism. Cardiolipin has been reported to be present in LDL39, suggesting that LDL and oxLDL could be a source of oxCL. However, a recent report did not confirm this finding40 and further research is needed to
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clarify this issue. We recently reported that antibodies against oxCL (anti-OxCL) but not anti-CL are protection markers for development of CVD15. One underlying mechanism could be that antiOxCL inhibits the effects of oxCL. It is not clear by what mechanisms and receptors oxCL exerts the proinflammatory effects described here. OxPL in general could interact with different types of
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receptors, also in a non-mutually exclusive way, including peroxisome proliferator-activated receptors (PPARs), the platelet-activating factor (PAF) receptor, and Toll-like receptors (TLRs)41
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but the oxCL-involvement of these remains to be shown. Our recent observations suggestes involvement of scavenger receptors15. We recently demonstrated that Annexin A5 is abundant in atherosclerotic lesions and that aCL can interfere with its binding to endothelial cells, promoting CVD in SLE12. Rand et al have demonstrated that Annexin A5 can form a crystalline shield over cell surfaces, which could have a protective function. However, this can be disrupted by aCL, causing the antiphospholipid antibody syndrome, characterized by arterial and venous thrombosis and also miscarriage42. Also, Annexin A5 has recently been reported to function as a potent antiatherothrombotic agent in a rabbit model of arterial thrombosis43. Mechanisms include interfering with tissue factor expression and by recovery of hypercoagulability
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. We recently reported that Annexin A5 improves endothelial
function, and decreases arterial inflammation and atherosclerosis in mouse models of 10
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atherosclerosis13. Further, the proinflammatory effects of a major phospholipid in the lesions, lysophosphatidylcholine, are inhibited by Annexin A5 and Annexin A5 inhibits uptake of oxLDL in macrophages, implying yet another antiatherosclerotic mechanism14. In line with this, Annexin A5 inhibits atherosclerosis development in a mouse model46. Here we report novel anti-inflammatory properties of Annexin A5, with potential relevance for inflammatory conditions including atherosclerosis and CVD. Annexin A5 inhibited the
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proinflammatory effects of oxCL, including induction of adhesion molecules in HUVEC, and LTB4 synthesis as well as calcium mobilization in human monocytes/macrophages and neutrophils. The mechanism is likely related to Annexin A5 binding to oxCL but not CL. Annexin A5 can thus in principle prevent these oxCL-induced effects by interactions with oxCL, though the precise
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molecular mechanism(s) remains to be elucidated.
Taken together, our findings indicate that oxCL can act as a novel pro-inflammatory factor,
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potentially causing or promoting atherosclerosis and plaque rupture in CVD and also playing a role in other chronic inflammatory conditions. Further, based on its capacity to inhibit oxCL-effects, we hypothesize that Annexin A5 could be developed into a therapeutic agent in atherothrombosis and plaque rupture and also in other inflammatory conditions were oxCL plays a role.
Acknowledgements
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This study was supported by the Swedish Heart Lung Foundation, the Swedish Research Council, the Stockholm County (ALF), the King Gustav V 80th Birthday Fund, CIDaT, Vinnova, AFA, Torsten Söderberg foundation, grants from the 6th Framework Program of the European Union, Priority 1: Life sciences, genomics and biotechnology for health (grant LSHM-CT-2006-037227
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CVDIMMUNE) with JF as coordinator. JF and AF are named as inventors on patent applications
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relating to Annexin A5.
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18 Tuominen, A., Miller, Y. I., Hansen, L. F., Kesaniemi, Y. A., Witztum, J. L. and Horkko, S., A natural antibody to oxidized cardiolipin binds to oxidized low-density lipoprotein, apoptotic cells, and atherosclerotic lesions, Arterioscler Thromb Vasc Biol, 2006, 26: 2096-2102. 19 Funk, C. D., Leukotriene modifiers as potential therapeutics for cardiovascular disease, Nat Rev Drug Discov, 2005, 4: 664-672. 20 Serezani, C. H., Aronoff, D. M., Jancar, S., Mancuso, P. and Peters-Golden, M., Leukotrienes enhance the bactericidal activity of alveolar macrophages against Klebsiella pneumoniae through the activation of NADPH oxidase, Blood, 2005, 106: 1067-1075. 21 Wan, M., Sabirsh, A., Wetterholm, A., Agerberth, B. and Haeggstrom, J. Z., Leukotriene B4 triggers release of the cathelicidin LL-37 from human neutrophils: novel lipid-peptide interactions in innate immune responses, Faseb J, 2007, 21: 2897-2905. 22 Goodarzi, K., Goodarzi, M., Tager, A. M., Luster, A. D. and von Andrian, U. H., Leukotriene B4 and BLT1 control cytotoxic effector T cell recruitment to inflamed tissues, Nat Immunol, 2003, 4: 965-973. 23 Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y. and Shimizu, T., A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis, Nature, 1997, 387: 620-624. 24 Yokomizo, T., Kato, K., Terawaki, K., Izumi, T. and Shimizu, T., A second leukotriene B(4) receptor, BLT2. A new therapeutic target in inflammation and immunological disorders, J Exp Med, 2000, 192: 421-432. 25 Helgadottir, A., Manolescu, A., Helgason, A., Thorleifsson, G., Thorsteinsdottir, U., Gudbjartsson, D. F., Gretarsdottir, S., Magnusson, K. P., Gudmundsson, G., Hicks, A., Jonsson, T., Grant, S. F., Sainz, J., O'Brien, S. J., Sveinbjornsdottir, S., Valdimarsson, E. M., Matthiasson, S. E., Levey, A. I., Abramson, J. L., Reilly, M. P., Vaccarino, V., Wolfe, M. L., Gudnason, V., Quyyumi, A. A., Topol, E. J., Rader, D. J., Thorgeirsson, G., Gulcher, J. R., Hakonarson, H., Kong, A. and Stefansson, K., A variant of the gene encoding leukotriene A4 hydrolase confers ethnicity-specific risk of myocardial infarction, Nat Genet, 2006, 38: 68-74. 26 Helgadottir, A., Manolescu, A., Thorleifsson, G., Gretarsdottir, S., Jonsdottir, H., Thorsteinsdottir, U., Samani, N. J., Gudmundsson, G., Grant, S. F., Thorgeirsson, G., Sveinbjornsdottir, S., Valdimarsson, E. M., Matthiasson, S. E., Johannsson, H., Gudmundsdottir, O., Gurney, M. E., Sainz, J., Thorhallsdottir, M., Andresdottir, M., Frigge, M. L., Topol, E. J., Kong, A., Gudnason, V., Hakonarson, H., Gulcher, J. R. and Stefansson, K., The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke, Nat Genet, 2004, 36: 233-239. 27 Dwyer, J. H., Allayee, H., Dwyer, K. M., Fan, J., Wu, H., Mar, R., Lusis, A. J. and Mehrabian, M., Arachidonate 5-lipoxygenase promoter genotype, dietary arachidonic acid, and atherosclerosis, N Engl J Med, 2004, 350: 29-37. 28 Spanbroek, R., Grabner, R., Lotzer, K., Hildner, M., Urbach, A., Ruhling, K., Moos, M. P., Kaiser, B., Cohnert, T. U., Wahlers, T., Zieske, A., Plenz, G., Robenek, H., Salbach, P., Kuhn, H., Radmark, O., Samuelsson, B. and Habenicht, A. J., Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis, Proc Natl Acad Sci U S A, 2003, 100: 1238-1243. 29 Qiu, H., Gabrielsen, A., Agardh, H. E., Wan, M., Wetterholm, A., Wong, C. H., Hedin, U., Swedenborg, J., Hansson, G. K., Samuelsson, B., Paulsson-Berne, G. and Haeggstrom, J. Z., Expression of 5-lipoxygenase and leukotriene A4 hydrolase in human atherosclerotic lesions correlates with symptoms of plaque instability, Proc Natl Acad Sci U S A, 2006, 103: 8161-8166. 30 Soehnlein, O., Multiple roles for neutrophils in atherosclerosis, Circ Res, 2012, 110: 875888. 31 Ionita, M. G., van den Borne, P., Catanzariti, L. M., Moll, F. L., de Vries, J. P., Pasterkamp, G., Vink, A. and de Kleijn, D. P., High neutrophil numbers in human carotid atherosclerotic plaques are associated with characteristics of rupture-prone lesions, Arterioscler Thromb Vasc Biol, 2010, 30: 1842-1848.
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32 Smith, W. L., Urade, Y. and Jakobsson, P. J., Enzymes of the cyclooxygenase pathways of prostanoid biosynthesis, Chem Rev, 2011, 111: 5821-5865. 33 Smyth, E. M., Grosser, T., Wang, M., Yu, Y. and FitzGerald, G. A., Prostanoids in health and disease, J Lipid Res, 2009, 50 Suppl: S423-428. 34 Ricciotti, E. and FitzGerald, G. A., Prostaglandins and inflammation, Arterioscler Thromb Vasc Biol, 2011, 31: 986-1000. 35 Frostegard, J., Ulfgren, A. K., Nyberg, P., Hedin, U., Swedenborg, J., Andersson, U. and Hansson, G. K., Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines, Atherosclerosis, 1999, 145: 33-43. 36 Febbraio, M., Podrez, E. A., Smith, J. D., Hajjar, D. P., Hazen, S. L., Hoff, H. F., Sharma, K. and Silverstein, R. L., Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice, J Clin Invest, 2000, 105: 1049-1056. 37 Iyer, S. S., Pulskens, W. P., Sadler, J. J., Butter, L. M., Teske, G. J., Ulland, T. K., Eisenbarth, S. C., Florquin, S., Flavell, R. A., Leemans, J. C. and Sutterwala, F. S., Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome, Proc Natl Acad Sci U S A, 2009, 106: 20388-20393. 38 Zheng, Y., Gardner, S. E. and Clarke, M. C., Cell death, damage-associated molecular patterns, and sterile inflammation in cardiovascular disease, Arterioscler Thromb Vasc Biol, 2011, 31: 2781-2786. 39 Deguchi, H., Fernandez, J. A., Hackeng, T. M., Banka, C. L. and Griffin, J. H., Cardiolipin is a normal component of human plasma lipoproteins, Proc Natl Acad Sci U S A, 2000, 97: 17431748. 40 Dashti, M., Kulik, W., Hoek, F., Veerman, E. C., Peppelenbosch, M. P. and Rezaee, F., A phospholipidomic analysis of all defined human plasma lipoproteins, Sci Rep, 2012, 1: 139. 41 Greig, F. H., Kennedy, S. and Spickett, C. M., Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation, Free Radic Biol Med, 2012, 52: 266-280. 42 Rand, J. H. and Wu, X. X., Antibody-mediated disruption of the annexin-V antithrombotic shield: a new mechanism for thrombosis in the antiphospholipid syndrome, Thromb Haemost, 1999, 82: 649-655. 43 Thiagarajan, P. and Benedict, C. R., Inhibition of arterial thrombosis by recombinant annexin V in a rabbit carotid artery injury model, Circulation, 1997, 96: 2339-2347. 44 Cederholm, A. and Frostegard, J., Annexin A5 in cardiovascular disease and systemic lupus erythematosus, Immunobiology, 2005, 210: 761-768. 45 Ishii, H., Hiraoka, M., Tanaka, A., Shimokado, K. and Yoshida, M., Recombinant Annexin2 inhibits the progress of diabetic nephropathy in a diabetic mouse model via recovery of hypercoagulability, Thromb Haemost, 2007, 97: 124-128. 46 Ewing, M. M., Karper, J. C., Sampietro, M. L., de Vries, M. R., Pettersson, K., Jukema, J. W. and Quax, P. H., Annexin A5 prevents post-interventional accelerated atherosclerosis development in a dose-dependent fashion in mice, Atherosclerosis, 2012, 221: 333-340.
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Figure 1
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Figure 1. oxCL-induced calcium mobilization. HMDMs (A) or PMNs (B) were seeded at density of 5 × 104 cells/well. HMDMs were incubated with medium containing 4 µM FURA-2AM plus 2.5 mM probenecid for 60 min or PMNs were incubated with medium with 4 µM FURA-2AM for 30 min in 5% CO2 at 37°C. After washing the plates were transferred to a fluorometer (Fluostar™,
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BMG Technologies), oxCL (a), oxCL+Annexin A5 (b), CL (c), Annexin A5 (d) or buffer solution were injected into individual wells, and the fluorescence intensities of the cells at 340 and 380 nm were monitored for 120 s. The results are given as the ratio of mean fluorescence intensities at 340
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Figure 2
Figure 2. Effect of oxCL on LTB4 production. HMDMs (A) or PMNs (B) were stimulated with CL (10 and 20 µg/ml), oxCL (10 and 20 µg/ml), or combined with annexin A5 (20 µg/ml) for 60 min (A) or 30 min (B). Buffer solution was added as negative control.
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Figure 3
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Figure 3. mRNA expression of 5-LOX, rather than COX-2 is increased by oxCL. HMDMs (1 × 106) were treated with control solution, CL (10 µg/ml) or oxCL (10 µg/ml) for 6 hr, afterwards the gene expression of 5-LOX and COX-2 were detected by real-time PCR (n = 4). *** P < 0,001, NS: no significance.
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Figure 4 A.
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B.
Figure 4. oxCL but not CL induces adhesion molecules in endothelial cells. (A) HUVECs were
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incubated with control solution (green filled), oxCL (10 µg/ml, red dotted) or CL (10 µg/ml blue filled). (B) HUVECs were incubated with control solution (green filled), oxCL (10 µg/ml, red dotted) or oxCL preincubated with Annexin A5 (5 µg/ml, purple filled) before stimulation. After 24
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h, the expression of ICAM-1 (CD54) and VCAM-1 (CD106) in HUVECs was detected by LSR
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Figure 5.
0.6
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OD value
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Annexin A5 concentration(µg/ml)
Figure 5. Binding of oxCL by Annexin A5. oxCL, CL and reduced CL (5 µg/ml) were coated on ELISA plates overnight. Annexin A5 (0, 0.32, 0.64, 1.28, 2.56, 5.12 and 10.24 µg/ml) were added
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and incubated for one hour.
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Cardiolipin (CL) is present in mitochondrial membranes but may be exposed during cell damage and is easily oxidized We demonstrate for the first time that Oxidized CL has inflammatory properties, inducing leukotriens, adhesion molecules and inflammatory enzymes Annexin A5 inhibits the proinflammatory effects of OxCL which could play a role in the antiatherosclerotic properties we previously reported
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S0021-9150(14)01163-0
DOI:
10.1016/j.atherosclerosis.2014.05.913
Reference:
ATH 13538
To appear in:
Atherosclerosis
Received Date: 4 September 2013 Revised Date:
25 April 2014
Accepted Date: 1 May 2014
Please cite this article as: Wan M, Hua X, Su J, Thiagarajan D, Frostegård AG, Haeggström JZ, Frostegård J, Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5., Atherosclerosis (2014), doi: 10.1016/j.atherosclerosis.2014.05.913. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5. Min Wan1*, Xiang Hua2*#, Jun Su2, Divya Thiagarajan2, Anna G Frostegård2, Jesper Z. Haeggström1 and Johan Frostegård2
1.
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1
Division of Physiological Chemistry II, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm, Sweden
IMM, Karolinska University Hospital, Huddinge, Karolinska Institutet, Stockholm, Sweden
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corresponding author
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#
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* contributed equally
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Objective
Cardiolipin (CL) is a phospholipid with an unusual dimeric structure containing four double-bonds and is easily oxidized. CL is present in mitochondria. Here we explored potential pro-inflammatory properties implicated in cardiovascular disease (CVD): activation of endothelial cells, 5lipoxygenase (5-LOX) and leukotriene B4 (LTB4), by oxidized CL (oxCL) and inhibitory effects of
Methods In
monocytes/macrophages
and
neutrophils,
calcium
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Annexin A5, an antithrombotic and antiinflammatory plasma protein.
mobilization
was
monitored
spectrophotometrically with Fura-2 and synthesis of LTB4 was analyzed by EIA. Expression of adhesion molecules on endothelial cells was studied by FACScan. Binding of Annexin A5 were
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analysed by ELISA. The mRNA expression of 5-LOX and cyclooxygenase-2 was assessed by RealTime PCR.
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Results
We demonstrate that oxCL but not its non-oxidized counterpart CL induces biosynthesis of LTB4 and increases intracellular concentrations of calcium in monocytes/macrophages and neutrophils. oxCL rather than CL selectively elevates gene expression of 5-LOX but not COX-2 in human macrophages. Furthermore, oxCL but not CL raises levels of adhesion molecules ICAM-1 and VCAM-1 in endothelial cells. Annexin A5 can bind oxCL to abolish all these oxCL-induced effects.
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Conclusions
oxCL may promote inflammation and related diseases especially in conditions involving unresolved apoptosis and necrosis, such as atherosclerosis, where free oxCL is likely to be released from liberated mitochondria. Increased intracellular calcium could activate 5-LOX to produce
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Leukotriene B4 (LTB4). Annexin A5 inhibits the pro-inflammatory effects of oxCL and its
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potential therapeutic use when oxCL is implicated in inflammation could be of interest.
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Introduction
Cardiolipin (CL) is a dimeric phospholipid which is known to be present in eucaryotic cells, bacteria and archaebacteria, however its functional role is only partly known1. It is more prevalent in cells with high metabolic activity, like heart and skeletal muscle, especially in mitochondrial membranes. The presence of CL in mitochondria and bacteria is interesting from an evolutionary point of view since mitochondria are likely to have a bacterial origin2.
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CL has a unique dimeric structure, highly enriched in linoleic acid moieties with double bonds, and susceptible to oxidation3, 4. It has been suggested that CL plays a role in generation of an electrochemical potential for substrate transport and ATP synthesis both in bacteria and mitochondria5, 6. CL that has undergone oxidation (oxCL) promotes delocalization and release of
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cytochrome c, predisposing to its release from mitochondria and the activation of cell death programmes4, 7, 8. Antibodies against CL (aCL) cause both venous and arterial thrombosis, and are known to be of major importance in rheumatic diseases, especially systemic lupus erythematosus
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(SLE), cardiovascular disease and venous thrombosis9.
Atherosclerosis, the major cause of CVD, is an inflammatory conditions, where macrophage- and endothelial cell activation are generally believed to play an important role. Also neutrophils have been discussed during recent years10. 5-LipoXygenase (5-LOX) has a calcium-binding domain, and increased intracellular calcium could activate 5-LOX to produce Leukotriene B4 (LTB4),
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which may play an important role in plaque rupture and other inflammation-related conditions11. Another important aspect of inflammation, in relation to atherosclerosis, is adhesion molecule expression on endothelial cells.
Annexin A5 is a member of the Annexin superfamily, and has anti-thrombotic properties due to
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interaction with phospholipids, especially phosphatidylserine, and thus inhibits the coagulation cascade. We recently demonstrated that antiCL suppressed binding of Annexin A5 to endothelial
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cells and suggested that Annexin A5 could have anti-atherothrombotic properties in SLE and in general12. In line with this Annexin A5 also has antiatherosclerotic properties, and modulates endothelial functions13, one mechanism could be inhibition of the major inflammatory phospholipid lysophosphatidylcholine14. We recently reported that enhanced levels of antibodies against oxCL (anti-OxCL), but not CL, are associated with an increased risk to develop CVD among 60-year olds in a population based study15 and that mortality among hemodialysis-patients where the risk of death from CVD is very high, is negatively associated with anti-OxCL16. Further, in SLE, where the risk of CVD is very high and the prevalence of atherosclerotic plaque is increased, we demonstrated a negative association between anti-OxCL and atherosclerotic plaques, including echolucent ones17. Our findings thus clearly indicate that oxCL as an antigen is present in humans, and also is
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generating immune responses among immune competent cells as B-cells. In principle oxCL could thus exert effects both in the atherosclerotic plaques and in the circulation or other tissues. OxCL is present in atherosclerotic lesions and apoptotic cells18 and is implicated in cell death4, 7, 8
, which is a major characteristic of atherosclerotic plaques. However, little is known about its
properties relevant to atherosclerosis and inflammation, which is the topic of the present study. We here report that oxCL in contrast to CL has pro-inflammatory properties, which could be inhibited
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atherosclerosis and its complications are discussed.
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by Annexin A5. The potential implications of these findings for inflammatory conditions including
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Materials and methods
Oxidation of CL CL from bovine heart was purchased as ethanol solution from Sigma (Sigma product C 1649) and was stored at –20° C. Hydro heart CL (reduced CL) was purchased from Avanti Polar Lipids, Inc. To generate saturated molecular species, CL was oxidized in aqueous solutions containing 1.5
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mmol/L tert-butylhydroperoxide and CuSO4 in 20 µmol/L, essentially as described. We have previously reported that CL treated according to this method was oxidatively modifed15.
Cell culture
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Human polymorphonuclear leukocytes (PMN)
Human PMNs were isolated from freshly prepared buffy coats (Karolinska Hospital blood bank,
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Stockholm, Sweden) by dextran sedimentation, hypotonic lysis of erythrocytes and gradient centrifugation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). PMNs were suspended at a density of 10 × 106/ml in Dulbecco’s PBS (Gibco (Invitrogen), Paisley, UK). PMN purity (> 95%) and viability (> 98%) was determined using Hemacolor (J.T. Baker, Utrecht, Holland) and Trypan Blue (Sigma Chemical Co.) staining, respectively.
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Human monocyte-derived macrophages (HMDM)
Human mononuclear cells were isolated from freshly prepared buffy coats (Karolinska Hospital blood bank, Stockholm, Sweden) by gradient centrifugation on Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden). Differentiation of human monocytes to macrophages was achieved
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by plating mononuclear cells in cell culture plates for two hours, and then unbound cells were washed away with PBS, followed by cell culture over seven days in RPMI-1640 medium with
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25mM Hepes, 1% L-glutamine, 1% penicillin-streptomycin and 10% FBS.
Endothelial cells
Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2 were purchased from Cascade Biologics, Inc (Portland, Ore). Cultures were maintained in EGMTM phenol red-free medium (Clonetics, San Diego, Calif), containing 2% of fetal bovine serum and supplements, at 37°C under humidified 5% CO2 conditions. All experiments were performed at passage 3 to 5. HUVECs were seeded at density of 6 × 104 cells/2 mL on 6-well plates (NUNC Inc, Naperville, Ill). After HUVECs attachment overnight, cells were ready for stimulation.
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Intracellular calcium mobilization
Human mononuclear leukocytes and PMNs were isolated from buffy coat as described above. Human monocytes were cultured and differentiated into human macrophages in black, 96-well plates with transparent bottom (Corning Costar; 5 × 105 cells/well) for seven days, and PMNs were seeded into black, 96-well plates with transparent bottom (Corning Costar; 5 × 104 cells/well), and spin down the plate at 120 × g for 5 min. When human macrophages and neutrophils were ready to
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use, the medium containing 4 µM FURA-2AM (Fura-2 acetoxymethyl ester), or buffer as appropriate was added into each well, and incubated for 30 min at 37°C and 5% CO2. Thereafter, cells were washed four times with 50 µl of a buffer solution (135 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 11 mM glucose, 11 mM Hepes, pH 7.4) before a final 50 µl volume of
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buffer was added to each well. Afterwards,the plates were transferred to a fluorometer (Fluostar™, BMG Technologies), and 50 µl of different agonists according to experimental designs or buffer
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solution as control was injected into individual wells and the fluorescence values in each well were monitored for the next 120 seconds. Control wells containing cells that had not been exposed to FURA-2AM were used to subtract background auto-fluorescence. The results are given as the ratio of mean fluorescence intensity (MFI) between 340 and 380 nm, and normalized by control.
Analysis of leukotriene B4 biosynthesis by EIA kit
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HMDMs (1 × 106) or PMNs (1 × 106) were incubated with different agents according to the experimental design, and then quenched with an equal volume methanol. After acidification to pH 3-4, the samples were purified by solid-phase extraction (SupelcleanTM LC-18, Supelco) and eluted in methanol. After evaporation of solvent under a stream of nitrogen, the samples were re-
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suspended in EIA buffer. The level of LTB4 was determined with LTB4 EIA kit (Cayman
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Chemical) by using dilutions within the linear portion of the standard curve.
Detection of adhesion molecules After the cells were washed with complete medium, oxCL and reduced CL (10 µg/ml) were added. For annexin A5 inhibition study, oxCL was incubated with Annexin A5 (5 µg/ml; Bender MedSystems GmbH, Austria) for 30 min before addition to cells. After 24 h incubation, detached floating cells were carefully collected, and remaining attached cells were gently harvested into Falcon FACS tubes after incubating with 400µl of FACSmax (Genlantis, USA) buffer for 10 min at room temperature. After centrifuging at 1200 rpm for 5 min, cells were resuspended in 50 µl Cell Staining Buffer (Biolegend, USA), incubated with FcR block (Miltenyi Biotec, Germany) for 10 min and then stained with PE-conjugated anti-CD54 (eBioscience) and FITC-conjugated antiHuman CD106 (Becton, Dickinson) for 30 min on ice. After washing with PBS, the fluorescence 6
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intensity of cells was measured by LSR Fortessa equipped with FACS Diva version 6 (BD Biosciences, San Jose, CA, USA).
Enzyme-Linked Immunosorbent Assay (ELISA) for Annexin A5 binding to antigen F96 microtiter polysorp plates (Nunc, Roskilde Denmark), were coated with oxCL, CL or Hydro Heart cardiolipin (R- CL) (Biosearch Technologies, Inc, Ca, USA) by incubation at a concentration
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of 5 µg/ml overnight at 40C. After five washings with PBS, the plates were blocked with 2% PBSBSA for 2 h at room temperature. Annexin A5 were added and incubated for another one hour. After washing, bound annexin A5 was detected by incubating subsequently with rabbit anti-human annexin V polyclonal antibodies (1:2000, Hyphen Biomed, Andresy, France) and polyclonal goat
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anti-rabbit Immunoglobulins/AP (1:3000, DakoCytomation). The reaction was developed with alkaline phosphatase substrate (Sigma), and optical density (OD) was read at 405 nm with an
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ELISA Multiscan Plus spectrophotometer (Molecular Devices Emax, San Francisco).
RNA extraction and quantitative real-Time PCR
Extraction of total RNA was performed by using the RNA Mini kit (Bioline, London, UK) including an on-column DNase digestion step. RNA concentration and quality were assessed by Nanodrop (Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized from total RNA by
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using SuperScript II reverse transcriptase (Life Technologies, Paisley, UK), and real-time PCR was performed in a TaqMan 7300 instrument (Life Technologies). Normalizations were made to β-actin. The primer/probe pairs were obtained by Assay-on-Demand (Life Technologies, Paisley, UK):
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respectively.
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human 5-LO, and β-actin with assay IDs Hs00386528_ml, Hs00153133_m1, and Hs99999903_ml,
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Results:
Intracellular calcium mobilization The influence of oxCL on intracellular calcium mobilization of HMDMs and PMNs were studied. Compared to control solution and CL, oxCL induces significantly increased intracellular calcium level in HMDM and PMNs (Fig. 1A and B). However, oxCL-induced intracellular calcium
change of intracellular calcium (Fig. 1A and B). LTB4 production
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mobilization is totally inhibited by Annexin A5, while Annexin A5 alone does not induce any
HMDMs and PMNs challenged with oxCL generate LTB4 (Fig. 2A, B) in a concentrationdependent manner. Native CL does not exhibit similar responses. LTB4 production induced by
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oxCL is completely blocked by Annexin A5 (Fig. 2A and B). 5-LOX gene expression in macrophages is upregulated by oxCL
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In addition to induction of a prompt LTB4 release from human neutrophils and macrophages, oxCL treatment for 6 h enhances the expression of 5-LOX mRNA, while CL exhibits no significant effect. However, oxCL treatment does not alter the level of COX-2 gene expression (Fig 3).
Effects of oxCL on endothelial cells
To study whether oxCL can promote the expression of adhesion molecules in endothelial cells,
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HUVECs were incubated for 24 h with oxCL or native CL. Our results indicate that oxCL significantly elevates both ICAM-1 and VCAM-1 expression, while native CL does not show the same effects (Fig.4A). Both the increased expression of ICAM-1 and VCAM-1 induced by oxCL
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were significantly inhibited by Annexin A5 (Fig. 4B).
Binding of Annexin A5 to cardiolipin
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To test whether Annexin A5 can bind various forms of cardiolipin, oxCL, CL and R-CL (5ug/ml) were coated on ELISA plates overnight before different concentrations of Annexin A5 were added to plates. Results show that binding of Annexin A5 in to oxCL is significantly stronger than binding of Annexin A5 to overnight air exposed CL. Moreover, no binding between reduced CL (R-CL) and Annexin A5 was detected (Fig. 5).
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Discussion
We report that oxCL but not CL has proinflammatory properties. OxCL could trigger LTB4 production in both human monocyte-derived macrophages (HMDM) and neutrophils. Further evidence of oxCL-mediated cell activation came from experiments demonstrating intracellular Ca2+ mobilization in these cell types. Leukotrienes are paracrine lipid mediators that have potent proinflammatory activities. Thus, LTB4 is one of the most potent chemotactic agents known to date,
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particularly for inflammatory cells and also evokes adhesion and transmigration of leukocytes across the endothelial barrier. LTB4 is biosynthesized from arachidonic acid by the sequential action of (5-LOX/FLAP) and LTA4 hydrolase, mainly in cells of myeloid lineage, such as neutrophils and macrophage19. Leukotrienes are known to exert broad pro-inflammatory effects as a
actions of LTB4
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part of the innate immune response and evidence is accumulating regarding the antimicrobial . More recently, however, the LTB4 signaling pathway was found to be
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important for linking early immune responses and the multiple classes of effector cells associated with acquired immunity22. Two G-protein coupled LTB4 receptors have been identified, BLT1 and BLT2, with high and low affinity for LTB4, respectively23, 24.
Our finding that oxCL can elicit LTB4 synthesis in macrophages is particularly interesting in the context of recent data implying LTB4 in atherosclerosis and CVD. Thus, a growing body of biochemical, histochemical and genetic data indicate that LTB4 plays an important role in the
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pathogenesis of human atherosclerosis and its sequelae, myocardial infarction and stroke25-28. Accordingly, mRNA levels for the three key proteins in LTB4 synthesis are significantly increased in human atherosclerotic plaque, and more pronounced in patients with ongoing symptoms of
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plaque instability29. Even though neutrophils are not commonly found in atherosclerotic lesions, recent findings indicate that they may still play a role, and are also detected in lesions using new techniques30. Interestingly, neutrophils accumulate at sites where plaque rupture are detected,
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suggesting a role in this major complication of atherosclerosis31. Interestingly, oxCL not only induces intracellular calcium mobilization resulting in a rapid LTB4 production, but also triggers further 5-LOX gene expression in human macrophages. In addition to 5-LOX, AA can be alternatively metabolized by cyclooxygenase (COX) coupled with metabolism by downstream enzymes into prostanoids32. The link of prostanoids and CVD has been widely documented33, 34. However, oxCL displays no effects on COX-2 gene expression in macrophages, which indicates that oxCL might selectively regulate the 5-LOX/leukotriene pathway. OxCL but not CL induced endothelial cells to express ICAM-1 and VCAM-1. Adhesion molecules play an important role to recruit monocytes into the intima in the artery, which is likely to be an early step in the inflammatory process that is characteristic of atherogenesis 10, 35. 9
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Also other evidence implies that oxCL could play a role in atherosclerosis and its complications. Even though the antigen recognized by a recently described monoclonal is not the same type of oxCL as used herein, it is still interesting to note that the apparently mildly oxidized form of CL was abundant in lesions18. In the oxidative environment present in lesions, it is possible that further oxidation of CL occurs, even though this is not studied here. Another recent observation is that oxCL competes with oxLDL for uptake into macrophages
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through scavenger receptors15. Such macrophages are believed to transform into foam cells, which are relatively inert, and constitute much of the cell debris in the atherosclerotic lesions and appear to promote atherogenesis10,
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. Another feature of atherosclerosis is apoptosis and necrosis, where
typically, apoptotic cells are not cleared properly, but instead appear to become necrotic. Dying
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cells activate the innate immune system and promote an inflammatory response with release of the proinflammatory cytokine IL-1ß, and the inflammasome37. According to the danger hypothesis,
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endogenous factors released during cell death such as high-mobility group protein B1 (HMGB-1), double-stranded DNA, amyloid-β-peptide and heat-shock proteins (HSP), could promote inflammation38. If mitochondria, leaking from abundant dead cells in atherosclerotic lesions or in other locations, liberate oxCL, this could be yet another pro-inflammatory mechanism. Cardiolipin has been reported to be present in LDL39, suggesting that LDL and oxLDL could be a source of oxCL. However, a recent report did not confirm this finding40 and further research is needed to
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clarify this issue. We recently reported that antibodies against oxCL (anti-OxCL) but not anti-CL are protection markers for development of CVD15. One underlying mechanism could be that antiOxCL inhibits the effects of oxCL. It is not clear by what mechanisms and receptors oxCL exerts the proinflammatory effects described here. OxPL in general could interact with different types of
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receptors, also in a non-mutually exclusive way, including peroxisome proliferator-activated receptors (PPARs), the platelet-activating factor (PAF) receptor, and Toll-like receptors (TLRs)41
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but the oxCL-involvement of these remains to be shown. Our recent observations suggestes involvement of scavenger receptors15. We recently demonstrated that Annexin A5 is abundant in atherosclerotic lesions and that aCL can interfere with its binding to endothelial cells, promoting CVD in SLE12. Rand et al have demonstrated that Annexin A5 can form a crystalline shield over cell surfaces, which could have a protective function. However, this can be disrupted by aCL, causing the antiphospholipid antibody syndrome, characterized by arterial and venous thrombosis and also miscarriage42. Also, Annexin A5 has recently been reported to function as a potent antiatherothrombotic agent in a rabbit model of arterial thrombosis43. Mechanisms include interfering with tissue factor expression and by recovery of hypercoagulability
43-45
. We recently reported that Annexin A5 improves endothelial
function, and decreases arterial inflammation and atherosclerosis in mouse models of 10
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atherosclerosis13. Further, the proinflammatory effects of a major phospholipid in the lesions, lysophosphatidylcholine, are inhibited by Annexin A5 and Annexin A5 inhibits uptake of oxLDL in macrophages, implying yet another antiatherosclerotic mechanism14. In line with this, Annexin A5 inhibits atherosclerosis development in a mouse model46. Here we report novel anti-inflammatory properties of Annexin A5, with potential relevance for inflammatory conditions including atherosclerosis and CVD. Annexin A5 inhibited the
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proinflammatory effects of oxCL, including induction of adhesion molecules in HUVEC, and LTB4 synthesis as well as calcium mobilization in human monocytes/macrophages and neutrophils. The mechanism is likely related to Annexin A5 binding to oxCL but not CL. Annexin A5 can thus in principle prevent these oxCL-induced effects by interactions with oxCL, though the precise
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molecular mechanism(s) remains to be elucidated.
Taken together, our findings indicate that oxCL can act as a novel pro-inflammatory factor,
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potentially causing or promoting atherosclerosis and plaque rupture in CVD and also playing a role in other chronic inflammatory conditions. Further, based on its capacity to inhibit oxCL-effects, we hypothesize that Annexin A5 could be developed into a therapeutic agent in atherothrombosis and plaque rupture and also in other inflammatory conditions were oxCL plays a role.
Acknowledgements
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This study was supported by the Swedish Heart Lung Foundation, the Swedish Research Council, the Stockholm County (ALF), the King Gustav V 80th Birthday Fund, CIDaT, Vinnova, AFA, Torsten Söderberg foundation, grants from the 6th Framework Program of the European Union, Priority 1: Life sciences, genomics and biotechnology for health (grant LSHM-CT-2006-037227
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CVDIMMUNE) with JF as coordinator. JF and AF are named as inventors on patent applications
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relating to Annexin A5.
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32 Smith, W. L., Urade, Y. and Jakobsson, P. J., Enzymes of the cyclooxygenase pathways of prostanoid biosynthesis, Chem Rev, 2011, 111: 5821-5865. 33 Smyth, E. M., Grosser, T., Wang, M., Yu, Y. and FitzGerald, G. A., Prostanoids in health and disease, J Lipid Res, 2009, 50 Suppl: S423-428. 34 Ricciotti, E. and FitzGerald, G. A., Prostaglandins and inflammation, Arterioscler Thromb Vasc Biol, 2011, 31: 986-1000. 35 Frostegard, J., Ulfgren, A. K., Nyberg, P., Hedin, U., Swedenborg, J., Andersson, U. and Hansson, G. K., Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines, Atherosclerosis, 1999, 145: 33-43. 36 Febbraio, M., Podrez, E. A., Smith, J. D., Hajjar, D. P., Hazen, S. L., Hoff, H. F., Sharma, K. and Silverstein, R. L., Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice, J Clin Invest, 2000, 105: 1049-1056. 37 Iyer, S. S., Pulskens, W. P., Sadler, J. J., Butter, L. M., Teske, G. J., Ulland, T. K., Eisenbarth, S. C., Florquin, S., Flavell, R. A., Leemans, J. C. and Sutterwala, F. S., Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome, Proc Natl Acad Sci U S A, 2009, 106: 20388-20393. 38 Zheng, Y., Gardner, S. E. and Clarke, M. C., Cell death, damage-associated molecular patterns, and sterile inflammation in cardiovascular disease, Arterioscler Thromb Vasc Biol, 2011, 31: 2781-2786. 39 Deguchi, H., Fernandez, J. A., Hackeng, T. M., Banka, C. L. and Griffin, J. H., Cardiolipin is a normal component of human plasma lipoproteins, Proc Natl Acad Sci U S A, 2000, 97: 17431748. 40 Dashti, M., Kulik, W., Hoek, F., Veerman, E. C., Peppelenbosch, M. P. and Rezaee, F., A phospholipidomic analysis of all defined human plasma lipoproteins, Sci Rep, 2012, 1: 139. 41 Greig, F. H., Kennedy, S. and Spickett, C. M., Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation, Free Radic Biol Med, 2012, 52: 266-280. 42 Rand, J. H. and Wu, X. X., Antibody-mediated disruption of the annexin-V antithrombotic shield: a new mechanism for thrombosis in the antiphospholipid syndrome, Thromb Haemost, 1999, 82: 649-655. 43 Thiagarajan, P. and Benedict, C. R., Inhibition of arterial thrombosis by recombinant annexin V in a rabbit carotid artery injury model, Circulation, 1997, 96: 2339-2347. 44 Cederholm, A. and Frostegard, J., Annexin A5 in cardiovascular disease and systemic lupus erythematosus, Immunobiology, 2005, 210: 761-768. 45 Ishii, H., Hiraoka, M., Tanaka, A., Shimokado, K. and Yoshida, M., Recombinant Annexin2 inhibits the progress of diabetic nephropathy in a diabetic mouse model via recovery of hypercoagulability, Thromb Haemost, 2007, 97: 124-128. 46 Ewing, M. M., Karper, J. C., Sampietro, M. L., de Vries, M. R., Pettersson, K., Jukema, J. W. and Quax, P. H., Annexin A5 prevents post-interventional accelerated atherosclerosis development in a dose-dependent fashion in mice, Atherosclerosis, 2012, 221: 333-340.
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Figure 1
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Figure 1. oxCL-induced calcium mobilization. HMDMs (A) or PMNs (B) were seeded at density of 5 × 104 cells/well. HMDMs were incubated with medium containing 4 µM FURA-2AM plus 2.5 mM probenecid for 60 min or PMNs were incubated with medium with 4 µM FURA-2AM for 30 min in 5% CO2 at 37°C. After washing the plates were transferred to a fluorometer (Fluostar™,
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BMG Technologies), oxCL (a), oxCL+Annexin A5 (b), CL (c), Annexin A5 (d) or buffer solution were injected into individual wells, and the fluorescence intensities of the cells at 340 and 380 nm were monitored for 120 s. The results are given as the ratio of mean fluorescence intensities at 340
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Figure 2
Figure 2. Effect of oxCL on LTB4 production. HMDMs (A) or PMNs (B) were stimulated with CL (10 and 20 µg/ml), oxCL (10 and 20 µg/ml), or combined with annexin A5 (20 µg/ml) for 60 min (A) or 30 min (B). Buffer solution was added as negative control.
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Figure 3
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Figure 3. mRNA expression of 5-LOX, rather than COX-2 is increased by oxCL. HMDMs (1 × 106) were treated with control solution, CL (10 µg/ml) or oxCL (10 µg/ml) for 6 hr, afterwards the gene expression of 5-LOX and COX-2 were detected by real-time PCR (n = 4). *** P < 0,001, NS: no significance.
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Figure 4 A.
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B.
Figure 4. oxCL but not CL induces adhesion molecules in endothelial cells. (A) HUVECs were
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incubated with control solution (green filled), oxCL (10 µg/ml, red dotted) or CL (10 µg/ml blue filled). (B) HUVECs were incubated with control solution (green filled), oxCL (10 µg/ml, red dotted) or oxCL preincubated with Annexin A5 (5 µg/ml, purple filled) before stimulation. After 24
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h, the expression of ICAM-1 (CD54) and VCAM-1 (CD106) in HUVECs was detected by LSR
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Figure 5.
0.6
0.4 0.3
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0.2 0.1 0.0 0.32
0.64
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2.56
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Figure 5. Binding of oxCL by Annexin A5. oxCL, CL and reduced CL (5 µg/ml) were coated on ELISA plates overnight. Annexin A5 (0, 0.32, 0.64, 1.28, 2.56, 5.12 and 10.24 µg/ml) were added
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Cardiolipin (CL) is present in mitochondrial membranes but may be exposed during cell damage and is easily oxidized We demonstrate for the first time that Oxidized CL has inflammatory properties, inducing leukotriens, adhesion molecules and inflammatory enzymes Annexin A5 inhibits the proinflammatory effects of OxCL which could play a role in the antiatherosclerotic properties we previously reported
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•