Phospholipolyzed LDL induces an inflammatory ... - The FASEB Journal

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Phospholipolyzed LDL induces an inflammatory response in endothelial cells through endoplasmic reticulum stress signaling Sarah Gora,* Seraya Maouche,* Rajai Atout,* Kristell Wanherdrick,† Ge´rard Lambeau,‡ François Cambien,* Ewa Ninio,* and Sonia-Athina Karabina*,1 *INSERM UMRS 937 and †Plate-forme Post-Geńomique, Universite´ Pierre et Marie Curie UPMC-Paris 6, Faculte´ de Me´decine Pierre et Marie Curie, Paris, France; and ‡Institut de Pharmacologie Molećulaire et Cellulaire, UMR 6097, CNRS-Universite´ de Nice-Sophia-Antipolis, Valbonne, France Secreted phospholipases A2 (sPLA2s) are present in atherosclerotic plaques and are now considered novel attractive therapeutic targets and potential biomarkers as they contribute to the development of atherosclerosis through lipoprotein-dependent and independent mechanisms. We have previously shown that hGX-sPLA2-phospholipolyzed LDL (LDL-X) induces proinflammatory responses in human umbilical endothelial cells (HUVECs); here we explore the molecular mechanisms involved. Global transcriptional gene expression profiling of the response of endothelial cells exposed to either LDL or LDL-X revealed that LDL-X activates multiple distinct cellular pathways including the unfolded protein response (UPR). Mechanistic insight showed that LDL-X activates UPR through calcium depletion of intracellular stores, which in turn disturbs cytoskeleton organization. Treatment of HUVECs and aortic endothelial cells (HAECs) with LDL-X led to activation of all 3 proximal initiators of UPR: eIF-2␣, IRE1␣, and ATF6. In parallel, we observed a sustained phosphorylation of the p38 pathway resulting in the phosphorylation of AP-1 downstream targets. This was accompanied by significant production of the proinflammatory cytokines IL-6 and IL-8. Our study demonstrates that phospholipolyzed LDL uses a range of molecular pathways including UPR to initiate endothelial cell perturbation and thus provides an LDL oxidation-independent mechanism for the initiation of vascular inflammation in atherosclerosis.—Gora, S., Maouche, S., Atout, R., Wanherdrick, K., Lambeau, G., Cambien, F., Ninio, E., Karabina, S.-A. Phospholipolyzed LDL induces an inflammatory response in endothelial cells through endoplasmic reticulum stress signaling. FASEB J. 24, 3284 –3297 (2010). www.fasebj.org

ABSTRACT

Key Words: unfolded protein response 䡠 inflammation 䡠 secreted phospholipase A2 group X 䡠 hGX sPLA2 䡠 modified LDL 䡠 atherosclerosis

phospholipids to generate lysophospholipids and free fatty acids, which are important inflammatory lipid mediators in atherosclerosis (1). The group IIA, III, V, and X (GIIA, GIII, GV, and GX, respectively) enzymes have been detected in human and/or mouse atherosclerotic lesions (2–5); however, the specific role of each enzyme remains to be elucidated. In contrast to other sPLA2s, GX sPLA2 is a neutral protein with the highest hydrolyzing activity toward phosphatidylcholine (PC), the major phospholipid component of lipoproteins and cell membranes (6). Recent studies using GX-deficient mice have implicated this enzyme in myocardial ischemia-reperfusion injury (7), allergeninduced airway inflammation (8), and lung injury (9), suggesting a prominent role of GX sPLA2 in inflammatory diseases. Endothelial cells being strategically located between blood and vessel wall play an important role in all stages of atherosclerosis. Normal endothelium provides vascular homeostasis, maintains blood fluidity, and controls vessel wall permeability, whereas an inflammatory environment activates endothelial cells to increase local blood flow and recruitment of circulating leukocytes to the damaged area (10). Accumulating evidence suggests that, apart from low-density lipoprotein (LDL) oxidation, other forms of LDL modification such as hydrolysis by sPLA2 (phospholipolysis) are potent endothelium activators (2). Conditions that induce cellular stress, such as hypoxia, protein glycation, and changes in calcium homeostasis, were shown to be causally related to atherosclerosis (11). The presence of stress in the cells is signaled by a set of intracellular signaling pathways collectively known as the unfolded protein response (UPR), and these pathways are now considered to play important roles in the initiation of inflammation in various diseases (12). Three endoplasmic reticulum (ER)-localized proteins initiate the UPR signaling cas1

Secreted phospholipases A2 (sPLA2s) represent a diverse family of structurally related calcium-dependent enzymes that hydrolyze the sn-2 position of glycero3284

Correspondence: INSERM U937, Faculte´ de Me´decine Pitie´-Salpeˆtrie`re, 91 Blvd. de l’Hoˆpital, Paris, 75634 Cedex 13, France. E-mail: [email protected] doi: 10.1096/fj.09-146852 0892-6638/10/0024-3284 © FASEB

cades: inositol-requiring 1␣ (IRE1␣), double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6) (13, 14). In resting cells, these sensors are maintained in an inactive state mainly through association with the ER molecular chaperone BiP (GPR78/HSPA5). The strength and duration of the stress in the ER (ER stress) determine how the cell alters its transcriptional and translational programs, in order either to survive (adaptive response) or to die (apoptosis), although the mechanisms underlying the transition between these 2 opposite outcomes are not yet fully understood. ER stress occurs at all stages of atherosclerotic lesion development in Apo E (⫺/⫺) mice (15) and has been associated with acute coronary syndrome (16). We have previously shown that human GX (hGX) sPLA2 is expressed in human atherosclerotic lesions and that in vitro hydrolysis of LDL by hGX sPLA2 results in a proinflammatory phospholipolysed lipoprotein particle (LDL-X), which induces human macrophage foam cell formation in vitro and the expression of adhesion molecules on the surface of human umbilical vein endothelial cells (HUVECs) (2). To gain more insight into the molecular mechanisms by which LDL-X induces endothelial cell activation, we have undertaken a transcriptomics approach employing endothelial cells treated with phospholipolyzed LDL vs. mock-treated LDL. Combining gene ontology (GO) enrichment and pathway analysis, we identified major stress pathways upregulated by LDL-X, with the UPR being prevalent. We show here for the first time that phospholipolyzed LDL, devoid of oxidative modification, can orchestrate the transcriptional activation of multiple genes mediated by the major UPR transducers eIF-2␣, IRE1␣, and ATF6. We provide mechanistic evidence that phospholipolyzed LDL induces calcium depletion of the ER which results in turn in UPR activation, cytoskeleton reorganization, activation of the p38 MAPK pathway, and release of proinflammatory cytokines. As the UPR alters the balance between cell survival and apoptosis, it plays an important role in the outcome of cardiovascular disease.

LDL isolation and treatment with sPLA2s LDL (d⫽1.019 –1.063 g/ml) was isolated from frozen plasma containing EDTA by density gradient ultracentrifugation, as described previously (18) and was free of HDL as detected by agarose gel electrophoresis and laser nephelometry for apoA1 (⬍0.05 g/L). Its protein content was determined by the bicinchoninic acid method (Pierce; Thermo Fisher Scientific, Cergy Pontoise, France). Freshly prepared and filtered (0.45 ␮m) sterile LDL, 1 mg of protein/ml in buffer containing 1 mM CaCl2, 12.5 mM Tris-HCl (pH 8.0), 0.25 M NaCl, and 0.0125% BSA was incubated the next day with 100 nM hGX sPLA2 for 3 h at 37°C and denoted as LDL-X. The reaction was stopped with 5 mM EDTA, which we tested to be sufficient to block the enzymatic activity of hGX sPLA2. LDL treated as above, but devoid of hGX sPLA2, is denoted as LDL and served as control. In selected experiments, LDL treated with hGX sPLA2 was reisolated by an additional ultracentrifugation step and extensively dialyzed against PBS. In a set of selected experiments LDL (1 mg protein/ml) was treated as described above with either 100 nM hGIIA sPLA2, 100 nM hGV sPLA2, or 100 nM of the catalytically inactive H48Q hGX sPLA2 mutant (17, 19) for 3 h at 37°C. Treatment of LDL with hGX, hGIIA, hGV sPLA2, or mock treatment as well as the subsequent reisolation did not result in oxidative modification of the particle as determined by the lack of production of conjugated dienes at 234 nm, a typical index of oxidation (20). Cell culture HUVECs were isolated by the method of Jaffe et al. (21) and grown as described previously (2). Selected experiments were performed with human aortic endothelial cells (HAECs), which were grown in EGM2 complete medium (Lonza; Levallois, Perret, France). NEFA and LysoPC measurements The extend of LDL hydrolysis by hGX sPLA2 was determined in an aliquot of sPLA2-treated and mock treated-LDL using the NEFA-FS kit (Diasys Pole, Condom, France) for nonesterified free fatty acid quantification and the AZWELL LPC kit (Cosmobio, Tokyo, Japan) to measure LysoPC production. Treatment of 1 mg/ml LDL with 100 nM hGX sPLA2 for 3 h resulted the release of 633 ⫾ 14 (n⫽3) nmol of LysoPC and 435 ⫾ 78 (n⫽6) nmol of NEFA. Cytotoxicity assay

MATERIALS AND METHODS Materials and antibodies

Cytotoxicity in the presence of the different LDL treatments was performed using the MTT-based kit TOX-1 (SigmaAldrich, Fallavier, France). Reverse transcription and quantitative-PCR (RT-qPCR)

Antibodies against BiP, IRE1␣, eIF2␣, phospho-eIF2␣, p38, phospho-p38, CREB, phospho-CREB, cJun, and phospho-cJun were purchased from Cell Signaling Technology (Ozyme, SaintQuentin-en-Yvelines, France). Antibodies against ATF3, ATF6, ATF4 (CREB-2), and GAPDH were purchased from Santa Cruz Biotechnology (Tebu-Bio, Le Perray En Yvelines, France). Antibodies against tubulin and lamin B1 antibodies were obtained from Zymed (Invitrogen, Cergy Pontoise, France). The calcium chelator BAPTA-AM was purchased from Calbiochem (VWR International, Fontenay sous Bois, France). Human group IIA, (hGIIA), V (hGV), and X (hGX) sPLA2s were produced in E. coli as described previously (17). STRESS-INDUCED PATHWAYS BY HGX SPLA2 MODIFIED LDL

Confluent HUVECs or HAECs were changed to serum-free medium overnight prior to treatments for 1, 3, or 6 h for time kinetic experiments or for only 6 h for all other experiments with 100 ␮g/ml LDL or LDL-X, reisolated LDL (LDL re), reisolated LDL-X (LDL-X re), or 100 nM hGX sPLA2. In some experiments, the cells were preincubated with the calcium inhibitor BAPTA-AM (10 ␮M) 30 min before lipoprotein treatment. Total RNA was isolated using RNAeasy (Qiagen, Courtaboeuf, France) including a DNase step. One microgram of total RNA was reverse transcribed to cDNA using Superscript II RNase H reverse transcriptase and oligo 3285

dT 18 mer (Invitrogen). In all assays, 10 ng of cDNA from each sample was amplified using a standardized program (15 min Thermo start DNA Polymerase activation step at 95°C; 40 cycles of 30 s at 95°C and 1 min at 60°C; followed by 1 min step at 95°C and dissociation steps at 36 cycles of 30 s between 60 and 95°C) in a MX 3005 real-time PCR system (Stratagene; Agilent, Massy, France) with a Absolute SYBR Green mix (Thermo Fisher Scientific). mRNA expression data were normalized to the levels of ribosomal protein L13a, RPL13A (NM_012423.2) used as housekeeping gene. The relative level of expression between sample 1 (control) and sample 2 (treated) was calculated using the ⌬⌬Ct formula: 2⫺(Ct1⫺CtRPL1)⫺(Ct2⫺CtRPL2). Normalized Ct values were used to calculate the gene expression ratio between the LDL-X and LDL samples. The expression ratios were then subjected to a log2 transformation to produce fold change data. The majority of the primers used in our study were obtained from the Harvard Primer Bank (Boston, MA, USA; http://pga.mgh.harvard.edu/primerbank/) (22, 23) or designed using the Primer Express 2.0 software from Applied Biosystems (Courtaboeuf, France). XBP1 mRNA splicing Total RNA was isolated from treated HUVECs as described above. Human XBP1 cDNA was amplified by RT-PCR (Qiagen OneStep RT-PCR kit) using primers that flank the intron excised by IRE1␣ exonuclease activity. Primer sequences used to amplify human spliced XBP1 were 5⬘-CAGCGCTTGGGGATGGATGC-3⬘ and 5⬘-CCATGGGGAGATGTTCTGGA-3⬘, human GAPDH 5⬘-CAGTCTTCTGGGTGGCAGTGA-3⬘, and 5⬘-TGCACCACCAACTGCTTAGC-3⬘. The protocol used for the PCR was as follows: 94°C (5 min), 30 cycles of 94°C (30 s), 55°C (30 s), 72°C (1 min), and 72°C (5 min). RT-PCR products were analyzed on a 3% agarose gel and visualized with ethidium bromide. Isolation of nuclear and cytoplasmic proteins and Western blot analysis Confluent endothelial cells were incubated overnight in serum-free medium and subsequently were treated for the indicated time periods with either 100 ␮g/ml LDL or LDL-X, or 100 nM hGX sPLA2. Cells were lysed in (3⫻) Laemmli SDS sample buffer with 50 mM DTT and 0.01% bromphenol blue. Nuclear and cytoplasmic proteins were extracted using nuclear extraction kits from Panomics (Ozyme) following the manufacturer’s instructions. Equal amounts of protein were analyzed by 4 –12% SDS-PAGE. Proteins were transferred to PVDF membranes and incubations with primary and secondary antibodies followed the manufacturer’s instructions. Immunoblots were revealed using the enhanced chemiluminescence reagents (Pierce; Thermo Fisher Scientific) and visualized using a high-performance chemiluminescence film (GE Healthcare, Saclay, France). The density of immunoreactive bands was measured using the image software Image J (U.S. National Institutes of Health; http://rsbweb. nih.gov/ij/), and bands were normalized either to a housekeeping protein (tubulin or GAPDH) or to total protein.

spectrometric mode after exposure to either LDL or LDL-X. Selected experiments were performed without the addition of 1 mM CaCl2 and 0.5 mM MgCl2 in the buffer. Ca2⫹ concentrations were calculated using the Grynkiewicz formula (24). Immunofluorescence Confluent HUVEC monolayers grown on glass coverslips were serum deprived overnight and subsequently incubated with 100 ␮g/ml LDL or 100 ␮g/ml LDL-X for 6 h at 37°C. The monolayers were then washed with PBS, fixed in 10% formalin solution neutral buffered (Sigma-Aldrich) for 10 min at room temperature, washed twice, and incubated in PBS-5% BSA for 1 h. Incubation with primary antibodies (anti-ATF4, anti-ATF3, anti-ATF6) diluted at 1:100 was carried out in PBS-1% BSA for 1 h at room temperature (RT). Cover slips were subsequently washed 4 times in PBS-1% BSA and incubated with goat anti-rabbit Alexa Fluor 594 IgG secondary antibody (1:200 in PBS-1% BSA) and DAPI (0.5 mg/ml) for 1 h at RT. Cover slips were then washed in PBS and mounted in homemade Mowiol. To study changes in cytoskeleton, coverslips were incubated with the high-affinity probe for F-actin, Alexa Fluor 488-conjugated phalloidin (Molecular Probes; Invitrogen), for 60 min at RT in the presence of DAPI (0.5 mg/ml). Confocal analysis was performed on a Leica SP2-AOBS confocal microscope, and the images were taken using alternating mode to minimize the channel interference (Leica Confocal Software; Leica Microsystems, Wetzlar, Germany). Measurement of cytokines levels in culture media IL-6, IL-8, VEGF, TNF-␣, and GM-CSF levels in cell culture supernatants were determined either by commercially available ELISA kits (R&D Systems, Lille, France) and/or by Luminex technology following the manufacturer’s instructions. Gene silencing with siRNA HUVECs were seeded in 6-well plates 24 h prior to transfection, and siRNA experiments were performed using Stealth RNAi (Invitrogen) and as a tranfection reagent Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM serum-free medium, according to the manufacturer’s protocol. Transfection efficiency was validated using a fluorescein-labeled dsRNA oligomer (BLOCK-iT Fluorescent Oligo; Invitrogen), which was transfected to cells also using Lipofectamine RNAiMAX. Cells were transfected with Stealth siRNA (Invitrogen, SARL) or low GC% (LGC), which served as a negative control. Lipofectamine RNAiMAX was diluted 1:100 with 10 nM duplex siRNA and incubated for 30 min at RT before addition to the cells. Following an incubation of 6 h, the transfection medium was replaced with fresh serum-free medium, and the cells were incubated with LDL or LDL-X for an additional 6 h. The efficacy of gene silencing was determined by qPCR and Western blot analysis. Microarray hybridization and raw data extraction

Determination of intracellular [Ca2ⴙ] levels Free intracellular Ca2⫹ concentration was measured in nonadherent HUVECs (4⫻106 cells) in the presence of the Ca2⫹-sensitive dye Fura-2 and pluronic acid (0.02%) at 37°C. Briefly, the measurements were performed in 2 ml HBSS buffer containing 1 mM CaCl2, 0.5 mM MgCl2, and 10 mM Hepes. The fluorescence was measured in a dual-wavelength 3286

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For the microarray experiment, HUVECs were isolated from 4 different cords (individuals) and treated with either 100 ␮g/ml LDL or LDL-X for 6 h. Experiments were carried out in duplicate using a dye-swap design. Total RNA was isolated using the RNA easy mini-column method (Qiagen). A DNase step was also included in order to make sure that there was no genomic DNA contamination. RNA quality (RIN⬎9, for RNA

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GORA ET AL.

Integrity Number) was determined using the Agilent 2100 Bioanalyzer with RNA 6000 Nano LabChip kit (Agilent Technologies, Massy, France), and quantities were determined using the NanoDrop microscale spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Probes were prepared from 200 ng total RNA, according to the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion; Applied Biosystems) except that volumes were divided by 2. First, double-stranded cDNAs were synthesized using the ArrayScript reverse transcriptase (Ambion kit) and a T7 oligo(dT) primer (Ambion kit). cDNAs afterward were used as a template for an in vitro transcription process. Amplified RNAs (aRNAs) were labeled by cyanine dye Cy3 or Cy5. aRNAs and labeled aRNAs qualities were both examined on the RNA Labchip (Agilent Technologies); their profiles were a distribution of sizes from 500 to 5000 nucleotides, with most of the aRNA, or labeled aRNA, at 1000 –1200 nucleotides. Cy3 and Cy5 incorporations were determined using the NanoDrop microscale spectrophotometer (NanoDrop Technologies) and were around 1 pmol/␮l for each dye. Hybridization and array image processing was carried out according to the manufacturer’s instructions (Agilent 60-mer oligo microarray processing protocol, version 7.0, G4140-90010; Agilent). Briefly, labeled RNAs from LDL and LDL-X samples (⬍0.75 ␮g per sample) were fragmented to an average size of 50 –100 nucleotides by heating the samples at 60°C and cohybridized overnight (17 h), at 60°C, to Agilent Human 1A (v2) oligo microarrays (Agilent) containing 22,575 features representing 17,244 distinct genes. After washing, microarrays were scanned on a Genepix 4000B scanner (Axon Instruments; Molecular Devices, France). Images processing and raw data extraction were performed using GenePix Pro software version 6.0 (Molecular Devices). Detailed description of protocols and all data have been submitted to the ArrayExpress repository (http://www.ebi.ac.uk/microarray-as/ae/) under accession number: E-MEXP-2256. Data preprocessing and analysis Preprocessing and statistical analyses were performed in R (25) and Bioconductor (http://www.bioconductor.org) environments. Fluorescence intensity values for Cy3 and Cy5 from each spot on the microarrays were imported into R. After filtering spots with bad flags, fluorescence intensity measurements (raw data) were background corrected using the normexp method (26). A within-array loess normalization followed by between-array quantile normalizations was performed. After correction for dye labeling effect, differential expression analysis was performed using the linear model for microarray data method (Limma) (27). A linear model was fitted to expression data for each genes, and an empirical Bayes modification of the t test (moderated t test) was used. To account for multiple hypotheses testing, the Benjamin and Hochberg multiple testing correction method was used (28). GO and pathway analyses Functional analysis based on the enrichment of GO (www. geneontology.org) categories in the list of differentially expressed genes was performed using the hyperGTest function of the GOstats R package (29) and ToppGene software (30). Tests were performed independently for biological process, molecular function, and cellular component GO classes. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) pathway analysis was carried out using WebGestalt software (31). STRESS-INDUCED PATHWAYS BY HGX SPLA2 MODIFIED LDL

Gene set enrichment analysis As a second analysis approach, gene set enrichment analysis (GSEA) was performed to determine whether an a priori defined gene set (groups of functionally related genes) shows a statistically significant difference between the 2 “phenotypes” (HUVECs treated with LDL vs. LDL-X). The first analysis approach (i.e., differential analysis followed by an over-/underrepresentation test of the list of differentially expressed genes) is limited by the statistical significance threshold used to select the list of genes differentially expressed by the treatment; however, it is well known that subtle changes in the expression levels of several genes within a pathway might be of biological relevance. To identify gene sets significantly enriched, GSEA was carried out using the SigPathway R package (32) on all genes (17,986). The gene sets tested were constructed from GO, KEGG, BioCarta, SABiosciences pathways, and literature databases using the Human Gene Sets GUI R package (unpublished results).

RESULTS Gene expression profile of HUVECs exposed to either LDL or LDL-X To investigate the molecular pathways leading to endothelial activation induced by LDL-X, we performed genome-wide transcriptional profiling of HUVECs exposed to either LDL or LDL-X. Treatment of endothelial cells for 6 h with LDL-X did not result in a significant cytotoxicity as compared to LDL-treated cells (absorbance units 0.132⫾0.06 for LDL-X vs. 0.140⫾0.03 for LDL, P⬎0.05). Microarray analysis revealed a significant modulation of several genes in LDL-X-treated cells. The changes in the expression of those genes are both statistically significant and quantitatively important (magnitude of change), as illustrated in Supplemental Fig. 1. Using as the threshold for gene list selection an adjusted value of P ⬍ 0.001 and a 2-fold change, we identified 255 probes corresponding to 219 distinct genes (143 overexpressed and 76 underexpressed in HUVECs exposed to LDL-X compared with those exposed to LDL). The list of those genes is provided in Supplemental Table 1. Pathway analysis demonstrated that LDL-X has a pleiotropic effect on HUVECs because several modulated genes are implicated in fundamental signaling pathways such as MAPK signaling, calcium signaling, and actin cytoskeleton (Supplemental Fig. 2). Furthermore, modulated genes were significantly (P⬍0.05) enriched in several GO categories including response to cell stress, cell cycle, proliferation, death, apoptosis, I-␬B kinase/ NF-␬B cascade, and the cellular response to unfolded protein (Supplemental Table 2). Several of these pathways will be addressed in detail below. Unfolded protein response In addition to the enrichment of GO categories related to cell stress and response to unfolded protein, the microarray analysis revealed that the major transcrip3287

tion factors and effectors of the UPR pathway were significantly up-regulated in LDL-X-activated endothelial cells (Supplemental Table 1). Time course experiments revealed that changes in gene expression of the major UPR components (DNAJB9, ASNS, ATF3, ATF4, XBP1, CEBPD, DUSP1) started 6 h after treatment of cells with LDL-X (Supplemental Fig. 3), except for HERPUD and DDIT3 genes, whose expression levels were significantly up-regulated after 3 h (P⬍0.05). UPR gene expression was concentration-dependent, with 100 ␮g/ml being the maximal LDL-X concentration tested (data not shown); in addition hGX sPLA2 added alone to cell cultures had no significant effect on UPR gene expression (Supplemental Fig. 4). LDL-X particles reisolated by ultracentrifugation followed by extensive dialysis were also able to induce UPR gene expression, indicating that the modified particle per se induces ER stress (Supplemental Fig. 4). To examine whether activation of UPR gene expression is specific for hGXtreated LDL in a set of selected experiments, we compared the ability of LDL treated for 3 h with 100 nM of either hGX, hGV, or hGIIA to induce UPR gene expression. As shown in Supplemental Fig. 5, only hGX-treated-LDL significantly induced the expression

of UPR genes. Similarly LDL treated with the catalytically inactive mutant H48Q did not significantly induce UPR gene expression, suggesting that the activity of the enzyme plays an important role in the cellular effects of LDL-X. Interestingly, in selected experiments in which endothelial cells were treated for 6 h with 5–25 ␮g/ml LysoPC, only the gene expression of ATF3 and DUSP1 was significantly induced (data not shown). Further experiments were performed only with LDL-X. In response to ER stress, the 3 ER-localized transmembrane signal transducers act in concert to regulate UPR through their respective signaling cascades. The protein chaperone BiP is considered as the master regulator for UPR activation. As shown in Fig. 1A, treatment of HUVECs with LDL-X resulted in a significant increase in the protein expression of BiP, a result that is consistent with the increased chaperone synthesis observed during UPR. Under normal conditions BiP binds to the luminal domains of IRE1␣, ATF6, and PERK, preventing their activation. Under stress, release of BiP from IRE1␣ allows IRE1␣ autodimerization and activation of its endoribonuclease activity. This leads to the catalytic removal of a 26-base intron from the mRNA of XBP1. This nonconventional splicing gener-

Figure 1. Activation of the IRE1␣-XBP1 branch of the UPR pathway by LDL-X. A, B) HUVECs were treated with either 100 ␮g/ml LDL or LDL-X for 6 h, and the induction of BiP (A) and IRE1␣ (B) was evaluated in total cell lysates by Western blot analysis. The density of immunoreactive bands was normalized to either GAPDH or tubulin, which were used as loading controls in the different experiments. *P ⬍ 0.05 vs. control. Results are representative of 4 experiments. C) RT-qPCR analysis of the total and spliced form of XBP1 in HUVECs treated with control (1), 100 ␮g/ml LDL (2), or LDL-X (3) for 6 h. Statistical analysis of treated cells (9 different HUVEC preparations) was performed using R. D) The presence of the spliced form of XBP1 (sXBP1) was further confirmed by RT-PCR using primers that specifically amplify the spliced form of XBP1 as described in Materials and Methods. Unspliced XBP1 is labeled uXBP1, and GAPDH was used as a housekeeping gene for the PCR. The results are representative of ⱖ3 experiments. 3288

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ates an active XBP1 transcription factor, known as spliced XBP1 (sXBP1). We confirmed an increased IRE1␣ protein expression in LDL-X activated HUVECs as compared to mock-LDL-treated cells (Fig. 1B). Using a set of specific primers that distinguish between total and the spliced form of XBP1 (33), we showed not only that the gene expression of total XBP1 is increased (Fig. 1C) in LDL-X-activated HUVECs, but also that the RNA undergoes splicing to generate active XBP1 (sXBP1) (Fig. 1D). Formation of sXBP1 is known to induce gene expression of the DnaJ family (34), and microarray analysis showed that the members DNAJB9 and DNAJB1 were significantly up-regulated in LDL-X activated HUVECs (confirmed also in separate experiments by qPCR; Supplemental Table 1). The second ER transmembrane component of the mammalian UPR is the basic region/leucine zipper transcription factor ATF6, which is expressed in a constitutive manner in its inactive form in the ER membrane. In response to ER stress a proteolytic cleavage of its N-terminal cytoplasmic domain occurs by the serine protease S2P to produce a potent transcriptional activator of chaperone genes (13). Using Western blot analysis we confirmed the increased expression of the active ATF6 form in the nucleus of the LDL-Xtreated cells (Fig. 2A). In addition, using immunofluoresence microscopy we detected ATF6 in the cytoplasm of LDL-treated and in the nucleus of LDL-X-treated endothelial cells (Fig. 2B). Genes whose expression is dependent on ATF6, such as XBP1 and CHOP/DDIT3, were found to be strongly up-regulated in LDL-Xtreated endothelial cells by both microarray analysis and qPCR (Supplemental Table 1). The third branch of the UPR regulates mainly the transcriptional control of protein synthesis. The ubiquitous ER transmembrane serine/threonine protein kinase PERK, when activated, phosphorylates the alpha subunit of the eukaryotic initiation factor 2, which, on the one hand, attenuates translation and thus reduces the protein folding load in the ER, but, on the other

hand, stimulates the selective transcription of genes including the transcription factor ATF4 (13). Western blot analysis of cell lysates from LDL-X-treated cells showed a significant phosphorylation of eIF2␣ as compared with mock-treated LDL or control cells (Fig. 3A). In addition, immunofluorescence microscopy showed a diffuse cytoplasmic localization of ATF4 in LDL-treated cells, whereas clusters of ATF4 were detected in the perinuclear and nuclear region in LDL-X-treated cells (Fig. 3B), indicating its activation. Microarray analysis showed that ATF4 and one of its target genes, the ASNS, involved in amino acid biosynthesis were significantly up-regulated in our conditions, and this was confirmed in separate experiments by qPCR (Supplemental Table 1). These results suggest that hGX-phospholipolyzed LDL activates all 3 branches of UPR in different types of endothelial cells (HUVECs and HAECs; Supplemental Table 1). The activation results in the up-regulation of a transcriptional program that aims to mediate the cell response to ER stress. Ca2ⴙefflux and actin reorganization As changes in Ca2⫹ homeostasis can trigger UPR (12) we next tested the hypothesis that LDL-X depletes intracellular Ca2⫹ stores. Using the fluorescent Ca2⫹sensitive probe Fura-2, we demonstrate that LDL-X induced a rapid and sustained rise in cytosolic Ca2⫹ levels (Fig. 4A). Mock-treated LDL also induced a rapid Ca2⫹ elevation, which, however, returned to baseline within seconds, suggesting that the signaling mechanisms leading to Ca2⫹ efflux are different between LDL and LDL-X. hGX sPLA2 alone (the same concentration as used to modify LDL) had no effect on Ca2⫹ efflux (data not shown). Selected experiments performed in the absence of Ca2⫹or Mg2⫹ in the buffer suggested that Ca2⫹ release originated from the intracellular pools. We next studied whether or not the increase in cytosolic Ca2⫹ observed in LDL-X-treated HUVECs

Figure 2. Activation of the ATF6 branch of the UPR pathway by LDL-X. A) HUVECs were treated with either 100 ␮g/ml LDL or LDL-X for 6 h, and the levels of p90ATF6 (top arrow) and p50ATF6 (bottom arrow) were evaluated by Western blot analysis in cytosolic and nuclear cell lysates as described in Materials and Methods. B) HUVECs were treated with either LDL or LDL-X for 6 h and subjected to immunofluorescence staining for ATF6 (red) as described in Materials and Methods. Nuclei were stained with DAPI (blue), and the merged images are shown. The results are representative of 3 experiments. Scale bar ⫽ 30 ␮m. STRESS-INDUCED PATHWAYS BY HGX SPLA2 MODIFIED LDL

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Figure 3. Activation of the PERK– eIF2␣ and ATF4 branch of the UPR pathway by LDL-X. A) HUVECs were treated with 100 ␮g/ml LDL or LDL-X for 6 h, and the phosphorylation of eIF2␣ was evaluated in total cell lysates by Western blot analysis. Levels of phospho-eIF2␣ were assessed as a ratio compared to total eIF2␣ using densitometric analysis. *P ⬍ 0.05 vs. control. Results are representative of 3 experiments. B) HUVECs were treated with either LDL or LDL-X for 6 h and subjected to immunofluorescence staining for ATF4 (red) as described in Materials and Methods. Nuclei were stained with DAPI (blue); merged images are shown. Results are representative of ⱖ3 experiments. Scale bar ⫽ 30 ␮m.

affects the organization of the cytoskeleton. The addition of LDL-X to the cells stimulated a massive formation of actin stress fibers, as shown by the specific staining of actin filaments with phalloidin (Fig. 4B); in contrast, LDL-treated or control cells do not show perturbations in their cytoskeleton organization. The effect of LDL-X on the organization of the cytoskeleton was further supported by the microarray data, which showed that the expression of connexin 37 (CON 37),

an important gap junction protein, was significantly down-regulated (log fold change ⫺1.106; adjusted P⬍0.0008 in LDL-X treated cells; confirmed by oPCR; see Supplemental Table 1). The change in connexin 37 expression in LDL-X-activated cells in parallel with the stress fiber formation strongly suggests that LDL-X modifies cellular organization and gap junction-mediated communication in the treated endothelial cells.

A

B

Figure 4. Ca2⫹ release and stress fiber formation in LDL-X-treated HUVECs. A) HUVECs in suspension were loaded with Fura-2 and stimulated with 100 ␮g/ml LDL (red line) or LDL-X (blue line). Intracellular Ca2⫹ levels were determined by the Fura-2 fluorescence. Arrow indicates lipoprotein addition. B) HUVECs were treated for 6 h with 100 ␮g/ml LDL or LDL-X, and then stained for F-actin with Alexa 488-phalloidin and counterstained with DAPI (nucleus) as described in Materials and Methods. Results are representative of 3 experiments. Scale bar ⫽ 10 ␮m. 3290

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To confirm that LDL-X-induced Ca2⫹ release triggers UPR activation, we pretreated HUVEC with the Ca2⫹ inhibitor BAPTA-AM, and we then observed a significant attenuation in gene expression of numerous

UPR-involved genes and in CON 37, as shown in Fig. 5. This result strongly suggests that the availability of intracellular Ca2⫹ is important in LDL-X induced UPR response and cytoskeleton reorganization.

Figure 5. Effect of BAPTA in UPR gene expression. Cells were preincubated with the Ca2⫹ chelator BAPTA-AM (10 ␮M) or vehicule (DMSO) for 30 min before treatment for 6 h with 100 ␮g/ml of LDL or LDL-X. Gene expression of major UPR transcription factors was examined by RT-qPCR. Statistical analysis of treated cells (4 different HUVEC preparations) was performed using R. Conditions 1–3 represent control, LDLtreated, and LDL-X-treated cells in the presence of DMSO, respectively; conditions 4 – 6 represent control, LDLtreated, and LDL-X-treated cells in the presence of BAPTA-AM, respectively. P values between LDL-X (BAPTA-AM) and LDL-X (DMSO) are indicated. STRESS-INDUCED PATHWAYS BY HGX SPLA2 MODIFIED LDL

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P38 activation The MAPK signaling pathways play an important role in the fate of cells in different biological processes (35) and are associated with ER stress and activation of the UPR (36). As genes for the MAPK signaling pathways were significantly enriched (P⬍0.05) in our microarray data (Supplemental Fig. 2), we examined possible changes in the phosphorylation status of the MAPK pathway in LDL-X-activated HUVECs. As shown in Fig. 6A, LDL-X triggered an early and sustained phosphorylation of the p38 branch of the MAPK, which started after 15 min of treatment and persisted up to 6 h. Mock-treated LDL or control cells showed a minimal phosphorylation of the p38. As the phosphorylation of p38 started within minutes, it is unlikely to be induced by UPR activation. Time-dependent experiments monitoring in parallel the activation of UPR sensors and the phosphorylation of p38 showed elF-2␣ phosphorylation, BiP, and IRE1␣ protein up-regulation started after ⱖ6 h of treatment of HUVECs with LDL-X, a result that correlated with the increased expression of UPR genes at the same time point, as shown in Supplemental Fig. 3, whereas the kinetics of the p38 as shown in Fig. 6A are completely different. Phosphorylation of p38 led to the phosphorylation of downstream effectors of the AP-1 family phospho-CREB and phospho-c-Jun in LDL-X-treated cells, as shown in Fig. 6B. Cytokine expression One of the gene sets significantly enriched in our microarray data represents cytokine-cytokine receptor interaction (Supplemental Fig. 2). Transcriptional differences for selected cytokine gene expression were confirmed using RT-qPCR (Supplemental Table 1), and their protein levels were evaluated using either the Luminex system and/or ELISA. As compared to cells treated with LDL, LDL-X-treated HUVECs secreted significantly more proinflammatory cytokine IL-8 and more VEGF. The levels of IL-6, although constantly higher in the supernatant of the LDL-X-treated cells, did not reach statistical significance. Cytokine expression remained significantly elevated when the cells were treated with the reisolated LDL-X particles, suggesting

again that the modified particle per se induces cytokine production (Supplemental Fig. 6). The protein levels of TNF-␣ and granulocyte/monocyte colony-stimulating factor, whose gene expression did not change under our microarray setting, were below the detection limit of the Luminex system (data not shown). Silencing of ATF3 Based on the significant up-regulation of the stressinducible transcription factor ATF3 in our microarray data, which was validated by qPCR (Supplemental Table 1), we hypothesized that LDL-X can trigger UPR in HUVECs via ATF3 activation. ATF3 activation was confirmed by Western blot analysis (Fig. 7A) and immunofluoresence, by which ATF3 was found in the nucleus of LDL-X-treated cells. In contrast, in LDLtreated cells ATF3 was mainly localized in the cytoplasm (Fig. 7B). To determine the potential role of ATF3 in LDL-X-induced UPR gene expression, we used a siRNA approach that effectively reduced endogenous ATF3 mRNA expression (Fig. 7D) and protein levels in both untreated and LDL-X-treated cells (Fig. 7C). However, the effects of ATF3 silencing were unexpected as the expression of known gene targets such as DDIT3/ CHOP was not significantly changed (Fig. 7D). The expression of the transcription factors DUSP1 and HERPUD, both involved in the UPR, was down-regulated in the presence of ATF3 siRNA (Fig. 7D), yet statistical significance for HERPUD was borderline (P⫽0.07). Although the absolute levels of expression of several UPR genes were decreased in the presence of ATF3 siRNA, their expression remained inducible by LDL-X (data not shown), suggesting that transcription of these genes is not strictly controlled by ATF3 but involves additional transcription factors.

DISCUSSION Atherosclerosis is characterized by the conjunction of inflammatory signals and accumulation of lipids leading to endothelial dysfunction and foam cell formation because of the uptake of modified LDL in the arterial wall (37, 38). We have previously shown that hGX

Figure 6. LDL-X induces p38 MAPK pathway activation. A) HUVECs were treated with 100 ␮g/ml LDL or LDL-X for the indicated times, and phosphorylation of p38 MAPK was evaluated by Western blot analysis in total cell lysates and compared to levels of total p38 protein. Results are representative of ⱖ3 different experiments in different gels. B) Cells were treated with 100 ␮g/ml LDL or LDL-X for 6 h, and the total and phosphorylated forms of CREB (left panel) and cJUN (right panel) were evaluated by Western blot analysis in total cell lysates. Results are representative of ⱖ3 experiments. 3292

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Figure 7. ATF3 silencing decreases LDL-X-induced UPR gene expression. A) HUVECs were treated with either 100 ␮g/ml LDL or LDL-X for 6 h and the activation of ATF3, i.e., its translocation to the nucleus, was evaluated in cytoplasmic and nuclear cell lysates by Western blot analysis. B) Density of the immunoreactive bands was normalized to the cytoplasmic protein tubulin for cytoplasmic lysates or to the nuclear protein lamin for nuclear lysates and visualized by immunofluorescence with an antibody against ATF3 (red) as described in Materials and Methods. Nuclei were stained with DAPI. Scale bar ⫽ 10 ␮m. Results are representative of ⱖ3 experiments. C) HUVECs were transfected with a siRNA against ATF3 or a negative control LCG siRNA, followed by treatment with 100 ␮g/ml LDL or LDL-X for 6 h. The silencing of ATF3 was validated by Western blot analysis in transfected cell lysates. D) Gene expression of ATF3, DDIT3, HERPUD, and DUSP1 was evaluated by RTqPCR in transfected cells. Results are means ⫾ sd of 3 different HUVEC preparations. *P ⬍ 0.05 vs. control; §P ⬍ 0.05 vs. LGC siRNA LDL-X.

sPLA2 is present in human atherosclerotic lesions, where it may encounter infiltrated LDL particles and may contribute to their hydrolysis. We have shown that such hydrolysis in vitro results in the formation of a proinflammatory LDL particle (LDL-X) that induces human macrophage foam cell formation and increases the expression of adhesion molecules on the surface of endothelial cells (2). The receptors implicated in the uptake of LDL-X by the vascular wall cells still remain unknown. In the current study we have explored the molecular mechanisms involved in the activation of endothelial cells by LDL-X. Using a global transcripSTRESS-INDUCED PATHWAYS BY HGX SPLA2 MODIFIED LDL

tomic approach we demonstrate here for the first time that phospholipolyzed LDL induces ER stress through multiple signaling pathways in endothelial cells. A set of genes significantly modulated in our microarray data attracted our attention because it consisted of several major transcription factors and effectors of UPR. UPR is a set of signaling pathways initiated when the cell encounters stressful conditions, such as nutrient deprivation, hypoxia, and oxidative stress, all of which may trigger ER stress. ER stress with the concomitant activation of UPR has been associated with a number of pathological conditions and has been recently linked to 3293

major cardiovascular events, including myocardial ischemia, heart failure (39), and accelerated atherosclerosis (15). ER stress markers are markedly increased in advanced atherosclerotic lesions in apo E (⫺/⫺) mice (15) and in humans, whereas their expression is weak in stable plaques and absent in control mammary arteries (40). Loading macrophages with cholesterol depletes ER Ca2⫹ stores, resulting in UPR activation and CHOP (DDIT3/GADD153)- induced apoptosis (41). In addition, oxidized LDL as well as bioactive lipid components of minimally modified or oxidized LDL (oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine, 7-ketocholesterol, and 4-hydroxynonenal) have been shown to activate ER stress sensors, causing inflammatory responses (33) and/or apoptosis in endothelial cells (40). Taken together, all these recent findings highlight the important role of lipids in inducing ER stress in vascular cells. ER stress alters the balance between survival and apoptotic signaling pathways and thus can dramatically alter the outcome of cardiovascular disease. In the present study we provide evidence that phospholipolysis can induce vascular inflammation through activation of the UPR response. In our setting, phospholipolyzed LDL is not oxidized, as demonstrated by the lack of production of conjugated dienes at 234 nm, and as discussed elsewhere (2, 42). Phospholipolyzed LDL induces UPR gene expression and protein phosphorylation much more rapidly and at a lower concentration when compared to oxidized LDL (40). Interestingly, under our experimental conditions, UPR activation was specific to hGX-treated LDL and did not occur when cells were exposed to hGIIA- or hGV-treated LDL (Supplemental Fig. 5). The hGV and hGX sPLA2 are up to 20 times more active than hGIIA sPLA2 in hydrolyzing LDL (6). In addition, hGX preferentially hydrolyzes arachidonate- and linoleate-containing PC species, whereas hGV hydrolyzes linoleate in preference to polyunsaturates. During evolution, hGIIA and hGV sPLA2 genes may have arisen after gene duplication (43), and thus it is possible that the structure and the active site of GV sPLA2 is more closely related to the narrower active site opening of GIIA than to GX sPLA2. This, in turn, may account for the relatively less effective hydrolysis of LDL-PC by GV sPLA2 (6). Moreover, as pointed out by Pan et al. (44), apart from the differences in the active site residues and the charged nature of the i-face of the sPLA2s, their substrate specificity may also be dependent on structural changes occurring on binding at the interface. Studies by Asatryn et al. (45) showed that LDL hydrolysis by phospholipases results in conformational changes in the LDL particle, suggesting resemblance to the in vivo circulating electronegative LDL. Finally, although hGX sPLA2 has not yet been detected in blood, one can argue that phospholipolysis of LDL by hGX may occur in plasma (in contrast to oxidation), and this phospholipolyzed LDL, in turn, can alter endothelial surface properties, initiating vascular inflammation. To exclude the effects of released lipid mediators in 3294

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UPR gene expression, selective experiments were performed with LDL-X particles reisolated by ultracentrifugation after the treatment with hGX sPLA2. Although a synergy between the modified LDL particle and the released lipid mediators in activating UPR cannot be excluded, the reisolated LDL had practically the same ability to induce UPR gene expression (Supplemental Fig. 4), suggesting that the modified particle per se has properties that induce ER stress and the UPR response. In selected experiments LDL treated with the hGX sPLA2 catalytic site mutant H48Q did not induce significant UPR gene expression (Supplemental Fig. 5), suggesting that the catalytic activity of the enzyme is important. On the other hand, when cells were treated with LysoPC, one of the major products of hGX action on LDL, we observed only increased expression of ATF3 and DUSP1 genes (data not shown), suggesting that the modification of the LDL particle is important for the UPR induction in addition to the effects of the hydrolysis products. These results are further supported by studies showing that arachidonic acid or lysophospholipids alone had little effect on UPR gene expression in endothelial cells (46). UPR activation was clearly demonstrated in LDL-X-treated endothelial cells as we detected increased synthesis of the protein chaperone BiP and increased production of IRE1␣, which was accompanied by the formation and nuclear localization of the active form of the transcription factor XBP1. In addition, in LDL-X-treated cells we observed increased phosphorylation of eIF-2␣ and nuclear localization of ATF4 and ATF6. The nuclear localization of all these transcription factors supports the idea that the cells are modulating their transcription program in order to cope with the LDL-X-induced stress. Indeed, the known target genes of ATF4, ATF6, and XBP1 were strongly up-regulated in LDL-X-treated cells. These included members of the DnaJ family known to facilitate protein folding and thus enabling reducing the load of unfolded proteins in the ER (34). In addition, we observed an increased transcription of ASNS, which ensures the supply of amino acids for protein biosynthesis, regulates the redox-balance, and may protect the cells against stress. The above data suggest that the LDL-X particles activated cells to induce a feedback mechanism in order to down-regulate the ER stress; however, we simultaneously observed an overexpression of the transcription factor CHOP mainly involved in apoptosis. These, at first glance, conflicting results can be rationalized by data suggesting that mild ER stress can activate all 3 sensors of the UPR response, but the stabilities of the prosurvival vs. the proapoptotic mRNAs and proteins will determine the cell fate. In addition, genetic and pharmacological experiments have shown that PERK-eIF-2␣ signaling and IRE1␣ can confer both pro- and antiapoptotic effects during ER stress (47– 49). All these data taken together suggest that in the experimental conditions we tested, proapoptotic and prosurvival mechanisms are still in equilibrium, in what might be “dormancy state”


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Figure 8. UPR signature and concomitant activation of multiple signaling pathways in LDL-Xtreated endothelial cells. Phospholipolyzed LDL through still unknown receptors induces multiple cellular stress pathways, which result in changes in calcium homeostasis, stress fiber formation, UPR activation, and proinflammatory cytokine secretion. In parallel, the p38 branch of the MAPK pathway is activated, enhancing the inflammatory response. Intensity and duration of the LDL-X-induced stress will define the fate of the cells.

(50). We are currently exploring the conditions that lead to either apoptosis or survival. The addition of LDL-X to endothelial cells resulted in sustained intracellular Ca2⫹ release, one of the earliest events observed in our experiments. Changes in Ca2⫹ homeostasis can profoundly alter vital cellular pathways and depletion of Ca2⫹ from the ER is associated with ER stress and UPR (51, 52). Pretreating endothelial cells with the Ca2⫹ inhibitor BAPTA before the addition of LDL-X resulted in a significant decrease in the expression of numerous UPR-involved genes. The pattern of Ca2⫹ efflux in the endothelial cells in the presence of LDL or LDL-X is different, suggesting implication of different signaling pathways and/or receptors. We are currently investigating the molecular mechanisms implicated. In addition, in LDL-X-treated cells we observed stress fiber formation and reorganization of the cytoskeleton, which was further confirmed by the down-regulation of connexin 37, an important gap junction protein whose expression level is an indication of endothelial dysfunction (53). In the presence of BAPTA, down-regulation of connexin 37 was abolished, indicating that intracellular Ca2⫹ is an important mediator of the effects observed in LDL-Xtreated cells. Recent work has demonstrated the activation of the p38-branch of the MAPK pathways under conditions of ER stress and during atherogenesis in vivo (54, 55). The activation of p38-MAPK has been linked to both proand antiapoptotic effects depending on the cellular environment and the apoptotic stimulus. Macrophage deficiency in p38a has been shown to promote apoptosis and plaque necrosis in advanced atherosclerotic lesions in mice by suppressing the Akt cell survival pathway (56). In our conditions the activation of the p38 pathway was rapid and sustained (from 15 min and up to 6 h treatment). Interestingly, the time course of p38 phosphorylation did not parallel UPR activation because phosphorylation of eIF-2␣, increased expresSTRESS-INDUCED PATHWAYS BY HGX SPLA2 MODIFIED LDL

sion of BiP and IRE1␣ (data not shown) and upregulation of the major UPR-related mRNAs were apparent after 6 h of treatment (Supplemental Fig. 3). These results strongly indicate that the cells initially try to cope with the stress and promote survival. However, at the end of the 6 h the apoptotic machinery is also activated. In conclusion, we report here for the first time that hGX sPLA2 phospholipolyzed LDL induces endothelial dysfunction through the activation of multiple stress-related pathways and induction of proinflammatory cytokines, leading either to apoptosis or to cell survival (Fig. 8). The cardiovascular outcome may depend on the balance between these opposite pathways. This study was supported by the Institut National de la Sante´ Et de la Recherche Me´dicale and by fellowships from the Fondation pour la Recherche Medicale (SPF20080512033) to S.A.K., (FDT200910244) to S.G., and (FDT20070910166) to S.M. The study was also supported by the European Union STREP Programme CVDIMMUNE (2006–2009) to E.N., by an ANR program (ANR 2006 Physiopathologie des maladies humaines: sPLA2/ATHEROSCLEROSE) to G.L. and E.N., and by the Centre National de la Recherche Scientifique (CNRS) and the Association pour la Recherche sur le Cancer to G.L. E.N. and G.L. are Directors of Research at CNRS. The authors acknowledge the excellent technical assistance of Monique Agrapart and Herve Durand.

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