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Proteomics, Metabolomics, and Immunomics on Microparticles Derived From Human Atherosclerotic Plaques

Originally publishedhttps://doi.org/10.1161/CIRCGENETICS.108.842849Circulation: Cardiovascular Genetics. 2009;2:379–388

Abstract

Background— Microparticles (MPs) with procoagulant activity are present in human atherosclerosis, but no detailed information is available on their composition.

Methods and Results— To obtain insights into the role of MPs in atherogenesis, MP proteins were identified by tandem mass spectrometry, metabolite profiles were determined by high-resolution nuclear magnetic resonance spectroscopy, and antibody reactivity was assessed against combinatorial antigen libraries. Plaque MPs expressed surface antigens consistent with their leukocyte origin, including major histocompatibility complex classes I and II, and induced a dose-dependent stimulatory effect on T-cell proliferation. Notably, taurine, the most abundant free organic acid in human neutrophils, which scavenges myeloperoxidase-catalyzed free radicals, was highly enriched in plaque MPs. Moreover, fluorescent labeling of proteins on the MP surface suggested immunoglobulins to be trapped inside, which was confirmed by flow cytometry analysis on permeabilized and nonpermeabilized plaque MPs. Colabeling for CD14 and IgG established that more than 90% of the IgG containing MPs were CD14+, indicating a macrophage origin. Screening against an antigen library revealed that the immunologic profiles of antibodies in MPs were similar to those found in plaques but differed profoundly from antibodies in plasma and unexpectedly, showed strong reactions with oligosaccharide antigens, in particular blood group antigen A.

Conclusions— This study provides the first evidence that immunoglobulins are present within MPs derived from plaque macrophages, that the portfolio of plaque antibodies is different from circulating antibodies in plasma, and that anticarbohydrate antibodies are retained in human atherosclerotic lesions.

Cell activation by agonists, physical, or chemical stresses stimulates bleb formation from the cell membrane, which is triggered by a rise of intracellular calcium and facilitated by modifications of the plasma membrane (ie, externalization of phosphatidylserine). From these blebs, virtually all cell types generate even smaller particles, termed microparticles (MPs). With a size of <1 μm, MPs were merely considered as a marker of cellular activation and damage, including apoptosis.1,2 However, MPs are also released in the circulation3 and ever since their potent procoagulatory properties were first recognized in the field of hemostasis,4–6 the interest in their potential pathophysiological importance has increased.7,8

Clinical Perspective on p 379

We have recently characterized MPs present in human atherosclerotic lesions.9 Notably, plaque MPs are derived from leukocytes (≈50%), erythrocytes, smooth muscle cells, and endothelial cells but not from platelets.9 They possess high tissue factor activity and expose phosphatidylserine, a major determinant of their procoagulant activity and their clearance by macrophages.10 However, no detailed information is currently available on the overall molecular composition. The aim of this study was to characterize MPs from human atherosclerotic plaques by using a combination of proteomic, metabolomic, and immunomic techniques.11 To analyze the protein composition, MP proteins were separated on large-format gradient gels and identified by tandem mass spectrometry (MS/MS). To investigate protein localization, MPs were surface labeled with CyDyes and separated by 2-dimensional gel electrophoresis (2-DE). The fluorescent tag allowed discriminating membrane-associated proteins from proteins within MPs. The proteomic analysis was complemented by high-resolution proton nuclear magnetic resonance spectroscopy to assess MP metabolites. Finally, the immunoglobulin portfolio of plaque MPs was screened against a combinatorial peptide and carbohydrate library to establish where the majority of antibody reactivity was found and then a sublibrary with defined antigens to partially identify the antigen specificity. We provide the first evidence that plaque MPs stimulate T-cell proliferation, that immunoglobulins are trapped within macrophage-derived MPs in human atherosclerosis, and that plaque antibodies show different antigen specificity compared with circulating plasma immunoglobulins and recognize carbohydrate antigens, including blood group antigen A.

Methods

Isolation of MPs From Atherosclerotic Plaques

MPs derived from human carotid atherosclerotic plaques were prepared as described previously.9 Surgical samples were rapidly rinsed in cold sterile phosphate-buffered saline solution supplemented with 100 U of streptomycin and 100 U/mL of penicillin, and atherosclerotic lesions were separated from the apparently healthy vessel wall. Plaques were then thoroughly minced using fine scissors in a volume of Dulbecco’s modified eagle medium (supplemented with 10 μg/mL of polymyxin B, 100 U streptomycin and 100 U/mL of penicillin and filtered on 0.22-μm membranes) corresponding to the respective weight of each lesion. The preparations were centrifuged first at 400g (15 minutes) and then at 12 500g (5 minutes) to remove cells and cell debris. The resulting supernatant was referred to as “plaque homogenate.” Part of the plaque homogenate was further centrifuged at 20 500g for 150 minutes at 4�C to pellet MPs.9 Pellets were gently suspended in fresh Dulbecco’s modified eagle medium (1/10 of volume corresponding to the respective weight of each lesion). Human atherosclerotic plaques (mean�SD, 713�70 mg) from 26 patients (72�2 years old, 69% men) undergoing carotid endarterectomy (CE) were included in the study, which was approved by the Hospital ethical committee. All patients gave their informed consent to the study. Indications for the CE were critical asymptomatic stenosis (>75%, n=13), stroke, or transient ischemic attack (n=13). Patients had common cardiovascular risk factors, such as hypertension (65%), diabetes (31%), hypercholesterolemia (62%), and smoking (42%). All patients were treated with antiaggregants. The cellular origin of plaque MPs was evaluated as reported before.9 One sample was used per subject.

1-DE-Liquid Chromatography-MS/MS

For proteomics, MPs of 3 patients were reconstituted in Laemmli buffer and separated by SDS-PAGE gels. After silver staining, gel bands were excised, subjected to in-gel tryptic digestion, and proteins identified by liquid chromatography (LC)-MS/MS (LCQ Deca XP Plus, Thermo Fisher). The outputs for each lane picked were combined in Scaffold (Proteome Software). The detailed methodology is available online.

Surface Labeling and 2-DE

Proteins on the MP surface were CyDye tagged using a modified protocol by Mayrhofer et al.12 In brief, CyDyes (GE Healthcare) were reconstituted in dimethylformamide, N,N-anhydrous, 99.8% (Aldrich) to make up stocks of 1 nmol/μL. These were then diluted in Hanks balanced salt solution (Gibco), pH 8.5, containing 1 mol/L urea to make up a 1 nmol/mL CyDye solution.13 MPs were labeled using the CyDye/urea solution. After 20 minutes, the reaction was stopped using 1 mL of L-Lysine (10 mmol/L, L8662, Sigma) for 15 minutes. Surface-labeled MPs were lysed in a complete lysis buffer (8 mol/L urea, 4% wt/vol CHAPS, 30 mmol/L TrisCl, pH 8.5). The separation by 2-DE involve adaptations of previously published protocols14,15 and is described online. Detailed protocols are provided on our website (http://www.vascular-proteomics.com).

Proton Nuclear Magnetic Resonance Spectroscopy

Metabolites were extracted from CE samples (n=4) and plaque MPs (n=4) as published previously16 and described online.

Flow Cytometry Analysis

Plaque homogenates were used for flow cytometry experiments. Labeling for immunoglobulin, apolipoprotein A1 (apoA1) and B (apoB) were performed before and after MPs permeabilization to determine surface versus intramicroparticle labeling. The detailed methodology is provided online.

Assessment of CD4+ T-Cell Proliferation

Blood mononuclear cells were isolated using Pancoll gradient of blood collected on heparin from patients undergoing endarterectomy. Negative selection of CD4+ cells was performed using an indirect magnetic labeling system for the isolation of CD4+ T cells from human PBMCs (CD4+ T-cell isolation kit II) on autoMACS columns (Miltenyi Biotec). CD4+ cells were pulsed with tritium and incubated with plaque MPs as described online.

Immunomics

For these experiments, MPs (20 500 g pellet), MP-depleted plaque homogenates and platelet-free plasma samples were obtained from 6 patients undergoing the CE. Venous blood was drawn at the time of surgery, and platelet-free plasma was prepared on 0.129 mol/L sodium citrate tubes as described previously.9 All samples were exposed to a PeptidePanel and GlycoPanel antigen library coated onto tiny aluminum particles with a unique barcode (Ultraplex, Pronostics, Cambridge, United Kingdom). The immunoreactivity of the samples was determined by labeling the bound antibodies with a fluorescent detection reagent in which different antibody subclasses were labeled with different colors. After mixing, the barcode of the particles with positive reactivity was read in a microscope-based reader (UltraPlex Smart Reader, Pronostics, Cambridge, United Kingdom), and the amount of label bound to each bead code was plotted providing an immunomic profile. Further details are provided online.

Statistical Analysis

Statistical analysis was performed using either the paired or unpaired Student t test. A P value of <0.05 was considered significant.

Results

Proteomics

MP proteins were separated by SDS-PAGE using large format gradient gels (4% to 12%). After silver staining, 64 bands (A1-H8, Figure 1) were excised and analyzed by LC-MS/MS (Figure 2) resulting in the identification of 151 unique proteins with a peptide probability >95.0%, a minimum of 2 peptides, and a protein probability >99.0%. A protein summary is provided in supplemental Table I. The identified peptides are listed in supplemental Table II. A functional classification of the identified proteins based on information from the Gene Ontology database returned the categories “membrane,” “extracellular matrix,” and “protein complexes” for cellular components (Figure 3A) and “metabolism” for biological processes (Figure 3B).

Figure 1. Protein separation by SDS-PAGE. Plaque-derived MPs were separated on large format gradient gels (4% to 12%) and stained with silver. Numbered bands (A1–H8) were excised and identified by LC-MS/MS. A summary of protein identifications is provided in supplemental Table I.

Figure 2. Tandem mass spectrometry (MS/MS). The product ion spectrum of the tryptic peptide GFGSDKEAILDIITSR was identified as annexin A6. All peptide identifications are provided in supplemental Table II.

Figure 3. Functional classification according to the Gene Onotology database. The proteins listed in supplemental Table I are shown as percentage of categories “cellular components” (A) and “biological processes” (B).

Membrane Proteins Reveal MP Origin

Consistent with their cellular origin,9 membrane proteins were predominantly derived from leukocytes, including CD14, CD36, CD11c (integrin α-X), CD18 (integrin β2), CD29 (integrin-β1), CD51 (integrin α-V), the B-cell receptor-associated protein 31, H-cadherin, and the vascular adhesion protein 1 (membrane copper amine oxidase). Smooth muscle cell (SM22-α) and erythrocyte markers (CD233) were also identified. In addition, plaque MPs contained the B2 bradykinin receptor and both subunits of the enzyme dolichyl-diphosphooligosaccharide-protein glycosyltransferase, also referred to as advanced glycosylation endproduct receptor-1. Notably, human leukocyte antigen class I and class II molecules were among the MP proteins. Validation by flow cytometry (n=12) confirmed that 15�2% of plaque MPs expressed major histocompatibility complex class II on their surface. Our observations, that MPs isolated from human atherosclerotic plaques express major histocompatibility complex class II together with potent costimulatory molecules such as CD40L,17 suggest that macrophage-derived MPs may contribute to lymphocyte activation within atherosclerotic lesions.18 A stimulatory effect was subsequently confirmed by incubating CD4+ T-lymphocytes with increasing concentrations of plaque MPs from the same patients (Figure 4, n=4).

Figure 4. Stimulatory effect of plaque MPs on T-cell proliferation. CD4+ T cells and plaque microparticles were obtained from the same patients. Note the proliferative response of T lymphocytes with increasing concentrations of plaque MPs. *Significant difference from supernatant (n=4), paired t test P<0.05.

Taurine Is the Most Abundant MP Metabolite

“Metabolism” emerged as the top category for biological processes in the Gene Ontology annotation. To obtain insights into the metabolite composition, we used proton nuclear magnetic resonance spectroscopy and compared plaque-derived MPs with CE samples. Quantitative data for water-soluble metabolites not present in the isolation medium are listed in supplemental Table III. Pie charts showing their relative abundance and representative nuclear magnetic resonance profiles are provided in Figure 5. Strikingly, taurine, the most abundant free organic acid in human neutrophils,19 which is implicated in the feedback inhibition of neutrophil/macrophage respiratory burst by scavenging myeloperoxidase-catalyzed free radicals,20 was highly enriched in plaque MPs compared with CE samples. MPs also contained high concentrations of lactate, the end product of anaerobic glycolysis, which is consistent with our proteomic findings that almost all glycolytic enzymes were present in plaque MPs, indicating that MPs might be still actively metabolizing subcellular entities. The smallest enrichment was observed for glycerophosphocholine, a degradation product of phosphatidylcholine, which was a major metabolite in the CE samples.

Figure 5. Proton nuclear magnetic resonance spectroscopy. Pie charts display the relative abundance of metabolites. Metabolite concentrations, their margins of error, and levels of statistical significance are given in supplemental Table III. Representative spectra of the CE (red) and MP (blue) samples are shown including a 4-fold enhanced image of the region 0.8 to 4.7 ppm in the upper panel for better visualization of the smaller peaks.

Surface Labeling Unravels Engulfment of Immunoglobulins

To address protein distribution in addition to composition, proteins on the MP surface were labeled with CyDyes before separation by 2-DE. Protein identifications are listed in supplemental Table IV. Only 2 proteins (marked with an asterisk) were found in the 2-DE but not the 1-DE-LC-MS/MS experiment. Strikingly, hemoglobin, apoA1, and vitronectin showed intensive fluorescence (Figure 6A, yellow boxes), but the heavy and light chains of immunoglobulins were not surface labeled despite their prominent silver staining (Figure 6B, red boxes). Although this dual labeling approach is only semiquantitative, it suggested that immunoglobulins might be trapped within plaque-derived MPs. Subsequent flow cytometry analysis confirmed this finding: the signal with fluorescein isothiocyanate conjugated anti-IgG antibodies was substantially stronger on permeabilized than on nonpermeabilized plaque MPs (Figure 7A and 7B). A similar 4-fold increase in the average number of positive events (permablized versus unpermeabilized) was obtained for Igkappa (Figure 7B) whereas the percentage of MPs staining for apoA1 was almost identical before and after permeabilization (Figure 7B), a finding consistent with the preferential surface labeling of apoA1 in our proteomic analysis. Interestingly, more MPs showed surface labeling for apoB than for apoA1 (3.6�1.0% versus 1.4�0.4%, n=10, t test P=0.039), although both apolipoproteins are present in similar concentrations in human plasma. Colabeling for CD14 and IgG revealed that almost all IgG containing MPs (93�7%) were CD14+, indicating a macrophage origin. However, only 31�13% of CD14+ MPs were double positive for IgG and surface CD14, whereas 69�13% stained for CD14 but not for IgG. Moreover, immunoglobulins were present in the MP-depleted supernatant even after filtration through a 0.1-μm filter indicating that immunoglobulins exist within macrophage-derived MPs and as free deposits in human atherosclerotic lesions (Figure 7C).

Figure 6. Surface labeling of plaque-derived MPs. Proteins on the MP surface were tagged with fluorescence dyes. After blocking the labeling reaction, MP proteins were separated by 2DE. Images were acquired using a fluorescence scanner (A). For total protein, counterstaining was performed with silver (B). Note that certain proteins are preferentially labeled with fluorescence (yellow boxes), ie, apoA1, whereas others show prominent silver staining, ie, immunoglobulins, but no surface labeling (red boxes). Numbered spots were identified by LC-MS/MS and are listed in supplemental Table IV.

Figure 7. Flow cytometry analysis of plaque-derived MPs. A, Representative graph for IgG labeling on permeabilized and nonpermeabilized plaque-derived plaque MPs. The shadowed peak represents labeling with corresponding isotype-fluorescein isothiocyanate and the white peak, labeling with anti-IgG antibody. B, Percentage of MPs positive for IgG, Igkappa, IgM, apoA1, and apoB. Full symbols represent nonpermeabilized and open symbols permabilized MPs. Note that the flow cytometry data are consistent with the proteomic data presented in Figure 6. C, Western blot analysis for IgG content. MP-depleted supernatants were obtained by centrifugation. MP-free supernatants were subjected to an additional filtration step to remove residual MPs. *Significant difference, t test P<0.05; ***P<0.001.

Antigen Specificity of Plaque Immunoglobulins

To examine whether the antibodies trapped within MPs were reactive and whether their specificity differed from immunoglobulins found in plaque and plasma, we used a novel bead-based technology to screen antibodies against a combinatorial antigen library. Surprisingly, most of the antibody reactivity within MPs was with carbohydrate but not peptide antigens. Further analyses were, therefore, directed against a sublibrary containing defined common carbohydrates to determine the antigen specificity of MP antibodies. For comparison, immunologic profiles were also obtained from the MP-depleted supernatant and from plasma samples of the same patients (n=6). Notably, although the immunologic profiles from plaque MPs (blue line) and the MP-depleted plaque homogenate (red line) were similar, plaque antibodies differed profoundly from plasma antibodies, which were profiled with and without an excess of calcium ions (Figure 8, green and black line, respectively). The addition of calcium did not change the antigenic profiles excluding calcium-dependent interactions. The plaque antibodies of all but 1 patient recognized the same antigen: blood group antigen A. IgG2, IgA, and IgM against the blood group antigen A are represented as peaks 10, 42, and 58 on the x-axis of the immunologic profiles (Figure 8A and 8B). Notably, the patient without reactivity in the plaque was blood group A positive (Figure 8C). Apart from antiblood group A, there were no additional major peaks in the IgG2, IgA, and IgD regions. The only other immunoglobulins detected were IgMs, some of which were directed against the Gal-α-(1,3)-Gal linkage, the antigen responsible for hyperacute rejection in xenotransplantation.21

Figure 8. Immunomic profiling of plaque and circulating antibodies. Plaque MPs, MP-depleted supernatant, and plasma were obtained from the same patients (n=6, supplemental Table V) and screened against a sublibrary, containing various carbohydrate antigens (supplemental Table VI). The antibody reactivity (y-axis) against antigens (x-axis) is plotted. Plasma antibodies were measured twice, once with and without the presence of extra Ca2+ (green and black line, respectively). Note that the antibody portfolio of pelleted MPs (blue line) and the MP-depleted supernatant (red line) is similar, but clearly distinct from plasma antibodies. The red arrows depict peaks with strong reactivity against the blood group antigen A. Examples are shown from 3 different patients: blood group O RhD positive (panel A), blood group O RhD negative (panel B), and blood group A RhD positive (panel C). The patient with blood group A had no detectable anti-A antibody signal in the plaque. The level of background fluorescence without antibody is ≈45 MFUs.

Discussion

A combination of “-omic” techniques was used to comprehensively analyze the MPs derived from human atherosclerotic lesions. The proteomic arm of the study identified membrane proteins confirming that plaque MPs stem primarily from leukocytes but also originate from smooth muscle cells and erythrocytes. The metabolomic approach revealed taurine as the most prominent metabolite in plaque-derived MPs further emphasizing the monocyte/neutrophil-created oxidative microenvironment in atherosclerotic plaques. The successful application of immunomic methods revealed that certain anticarbohydrate-moiety antibodies were enriched in the plaque and that a subpopulation of CD14+ MPs carried an intravesicular antibody load within atherosclerotic lesions. Our study provides the first evidence for an engulfment of immunologlobulins within MPs of plaque macrophages and partially unravels their antigen specificity. Thus, besides being an important determinant of plaque thrombogenicity, MPs might play a previously unrecognized role in modulating tissue inflammation as supported by their proliferative effect on CD4+ T lymphocytes.

Proteomics of MPs to Target Membrane Proteins

Of the different cellular subproteomes, those embedded in the plasma membrane are of substantial interest because they regulate key biological functions, but the physiochemical characteristics and low abundance render analysis by proteomics challenging.13 Although proteomics has been previously applied to atherosclerosis,22–24 none of these studies has successfully targeted the membrane subproteome. To overcome the obstacles of membrane proteomics in complex tissues, we used a novel approach by analyzing tissue-derived MPs. So far, proteomic studies have been published on MPs from plasma,25 platelets26 and cultivated endothelial cells,27 and T lymphocytes.28 However, MP generated in vitro are not necessarily representative for MPs present in vivo because the phenotype of the released MPs is dependent on the agonist used to activate the parent cells. Besides, plaque-derived MPs are a population of membrane blebs originating from the cells constituting the atherosclerotic lesion and reflect their biological complexity and heterogeneous composition.9 Thus, it is essential to complement the existing in vitro data sets by addressing the composition of in vivo-derived MPs. In this study, we were able to identify more than 150 proteins including membrane receptors of plaque-derived MP, despite the scarcity of the biological material available and the presence of high-abundant plasma proteins.

Immunoglobulins Are Contained Within Plaque MPs

Immunocomplexes resulting from the interaction of IgM natural antibodies with oxidized lipids are known to contribute to the removal of plaque antigens and prevent foam cell formation by blocking the uptake of oxidized lipids by plaque macrophages.29 However, in vitro studies indicate that other antibodies with different isotypes (eg, IgG) also bind oxidized lipids and contain Fc domains capable of binding to macrophage Fc receptors, which could actually promote the uptake of immune complexes and contribute to the formation of lipid-filled macrophage-derived foam cells.29 A potential proatherogenic role of IgG Fc receptors is supported by the protective effect of Fcγ deficiency in apoE-knockout mice.30 Our finding that IgG is trapped inside macrophage-derived MPs isolated from human atherosclerotic lesions provides additional evidence that IgG might be taken up by macrophage Fcγ receptors previously described in human atherosclerotic plaques.31 Although the presence of antibodies and complement components would support the existence of immune complexes, it is currently unclear to what extent the antibodies within plaque MPs are bound to complement/antigens or present as free immunoglobulins. This warrants further investigation in future studies because overloading plaque resident macrophages with immune complexes, oxidized lipids, or apoptotic cells could lead to macrophage apoptosis and trigger MP release within the lesion.

It has been shown previously that oxidized plasma lipoproteins (low-density lipoprotein [LDL] or high-density lipoprotein) are incorporated by macrophage scavenger receptors for subsequent lysosomal degradation and relocation of modified apoA1 and apoB to the plasma membrane.32 Moreover, CD4+ T cells reactive to oxidized LDL have been cloned from human lesions,33 and IgG and IgM antibodies that recognize epitopes of oxidized lipids are present in large amounts in advanced human atherosclerotic plaques.34–36 Our data add to these findings by demonstrating that plaque MPs express major histocompatibility complex on their surface and activate CD4+ T lymphocytes, which could contact and stimulate B lymphocytes, although scarce in atherosclerotic plaques, to produce immunoglobulins specific against plaque antigens. Notably, antigens, such as phosphatidylcholine, are found not only in oxidized LDL but also on apoptotic cell membranes,37 and therefore likely to be present on plaque MPs.

The Portfolio of Plaque Antibodies Differs From Plasma

Our immunomic experiments revealed that plaque antibodies react with other antigens apart from oxidized LDL: First, the antibodies trapped within MPs were still reactive as demonstrated unambiguously by their strong signal in the immunomic profiling experiment. Second, although there is no selective retention of plaque antibodies in MPs, their antigen specificity is clearly distinct from the antibodies circulating in plasma. These findings do not exclude a carryover of plasma antibodies into the MP preparations, but at least the capture of plasma antibodies within atherosclerotic plaques must highly specific. Third, although the plasma profiles varied greatly between patients, antibodies against the blood group antigen A were consistently detected in atherosclerotic plaques and these anti-A antibodies were of 3 heavy chain isotypes (IgG2, IgA, and IgM), providing additional evidence that this result is not an artifact. For unknown reasons, specific immune responses against many carbohydrates are preferentially IgG2, and the carbohydrate antigens recognized by IgG2 are present not only in several tumor cells but also in endothelial cells. Carbohydrate antigens of the ABO blood group are expressed at high levels on endothelium, probably attached to von Willebrand factor. The ABO blood group influences the rate of proteolysis of von Willebrand factor38 and increase its adhesive activity.39 In fact, there is a direct relationship between ABO genotype and the amount of A antigen expressed on circulating von Willebrand factor.40 The latter has been shown to increase the risk of ischemic stroke.41 Other antibodies detected in the atherosclerotic plaques were human IgM against Gal-α-(1,3)-Gal. This terminal carbohydrate epitope is the major target for natural antibodies to pig cells in humans and formed by the α-1,3 galactosyl transferase, which places a terminal galactose residue in an α-linkage to another galactose.42 These results extend our previous observation that the pattern of the anticarbohydrate immune response is different in plasma from patients with advanced atherosclerosis.43 We now provide the first evidence that besides antibodies to oxidized LDL,44 anticarbohydrate antibodies are also retained within human atherosclerotic lesions. Their presence, however, does not necessarily imply alterations in the amount or disposition of carbohydrate antigens but may reflect alterations in the immune system affecting the levels of natural antibodies to common environmental antigens.45–47

Limitations of the Study

Although proteomics and metabolomics have proven valuable tools to array authentic proteins and metabolites from human tissues, it is important to acknowledge that additional proteins and metabolites are likely tobe present in plaque MPs.17 Moreover, although this study identified anticarbohydrate immunoglobulins as a component of the plaque antibodies present, our findings do not exclude other antibody specificities within the portfolio, in particular against peptide antigens, which tend to be recognized as conformational rather than linear epitopes. In addition, lipids, modified lipid reactivities, and peptides with posttranslational modifications were not present in the combinatorial antigen library. Hence, antioxidized LDL antibodies, which are probably a major component of plaque MPs,48 would remain undetected by our assays.

Conclusion

To the best of our knowledge, this study is the first to investigate not only the protein composition but also the protein distribution, metabolite content, and immunologic profiles of MPs49 by using 3 state-of-the-art techniques. The comprehensiveness provided by proteomics, metabolomics, and immunomics revealed a novel role of MPs in inflammation, which is a key determinant of plaque stability and progression.

Sources of Funding

This work was funded by the European Vascular Genomics Network (LSHM-CT-2003-503254; Brussels, Belgium) and grants from the British Heart Foundation, Oak Fundation, Agence Nationale de la Recherche (ANR-06-Physio-038), and Leducq Foundation LINK project. Dr Boulanger is supported by a Contrat d'Interface INSERM-APHP and Dr Mayr by a Senior Research Fellowship of the British Heart Foundation.

Disclosures

Dr Grainger was Chief Scientific Officer at Pronostics.

Footnotes

Correspondence to Manuel Mayr, MD, PhD, Cardiovascular Division, King’s College London, 125 Coldharbour Lane, London SE5 9NU, United Kingdom. E-mail

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circcvgCirc Cardiovasc GenetCirculation: Cardiovascular GeneticsCirc Cardiovasc Genet1942-325X1942-3268Lippincott Williams & WilkinsCLINICAL PERSPECTIVE082009

Cell activation stimulates bleb formation from the cell membrane. From these blebs, virtually all cell types generate even smaller particles, which are termed microparticles (MPs). MPs with procoagulant activity are present in human atherosclerosis, but detailed information on their composition is not available. In the present investigation, we used a combination of “-omic” techniques to comprehensively analyze MPs derived from human atherosclerotic lesions. The proteomic arm of the study identified membrane proteins confirming that plaque MPs stem primarily from leukocytes but also originate from smooth muscle cells and erythrocytes. The metabolomic approach revealed taurine as the most prominent metabolite in plaque-derived MPs. Because taurine serves as a negative feedback after oxidative burst, these data further emphasize the presence of a monocyte/neutrophil-created oxidative microenvironment in atherosclerotic plaques. The successful application of immunomic methods revealed that certain anticarbohydrate-moiety antibodies were enriched in the plaque and that a subpopulation of CD14+ MPs carried an intravesicular antibody load within atherosclerotic lesions. Notably, carbohydrate antigens of the ABO blood group are expressed at high levels on endothelium, probably attached to von Willebrand factor. Most of the antibody reactivity with carbohydrate antigens were directed against the blood group antigen A and against the Gal-α-(1,3)-Gal linkage, the antigen responsible for hyperacute rejection in xenotransplantation. Our study provides the first evidence for an engulfment of immunologlobulins within MPs of plaque macrophages and provides clues about their antigen specificity. Thus, besides being an important determinant of plaque thrombogenicity, MPs might play a previously unrecognized role in modulating tissue inflammation as supported by their proliferative effect on CD4+ T lymphocytes.

The online-only Data Supplement is available at http://circgenetics.ahajournals.org/cgi/content/full/CIRCGENETICS.108.842849/DC1.

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