Published online before print June 22, 2006, doi: 10.1161/01.ATV.0000233333.07991.4a
© 2006 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
A Natural Antibody to Oxidized Cardiolipin Binds to Oxidized Low-Density Lipoprotein, Apoptotic Cells, and Atherosclerotic Lesions
From the Department of Internal Medicine and Biocenter Oulu (A.T., Y.A.K., S.H.), University of Oulu, Finland; Department of Medicine (Y.I.M., L.F.H., J.L.W., S.H.), University of California San Diego, San Diego, Calif.
Correspondence to Sohvi Hörkkö, University of California San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0682. E-mail shorkko{at}ucsd.edu
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Abstract |
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Objective— Cardiolipin (CL) is found in membranes of bacteria, in the inner membrane of mitochondria and in plasma low-density lipoprotein (LDL). Anticardiolipin antibodies (aCL) are associated with disease states, and we have suggested that many aCL bind to oxidized CL (oxCL) but not native CL. To determine the immunogenicity and origins of oxCL in vivo, we cloned a natural antibody to oxCL.
Methods and Results— A monoclonal IgM antibody to oxCL (LRO1) was cloned from a nonimmunized LDLR–/– mouse. The VH sequence originated from the VHGam3.8 germline with one nucleotide difference, and the V
was 100% identical to V19–20 germline gene, making LRO1 a natural antibody. LRO1 bound specifically to oxCL and oxidized-LDL, but not to native CL or native LDL. LRO1 epitopes were demonstrated in apoptotic, but not in viable, Jurkat cells by flow cytometry, immunofluorescence and deconvolution microscopy. Human and rabbit atherosclerotic lesions contained LRO1 epitopes. Human LDL (n=113) showed LRO1 immunoreactivity, which correlated with aCL IgG titers (r=0.32, P=0.0004).
Conclusions— These data demonstrate that some aCL antibodies are highly conserved natural antibodies binding to oxCL in oxLDL, apoptotic cells, and atherosclerotic lesions. This suggests that oxCL is one of the pathogen-associated molecular patterns of innate immunity and gives insight into the pathogenic events of diseases with increased titers of aCL antibodies.
A natural anticardiolipin antibody (LRO1) binding to oxidized cardiolipin (oxCL), oxidized LDL, apoptotic cells, and atherosclerotic lesions was cloned from a nonimmunized LDLR–/– mouse. These data demonstrate that oxCL is an important antigenic determinant in vivo and gives insight into the pathogenic events of diseases with increased titers of aCL antibodies.
Key Words: autoantibody • cardiolipin • lipoproteins • oxidized phospholipids
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Introduction |
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Antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies characterized by their reactivity to anionic phospholipids, typically cardiolipin (CL), phospholipid/protein complexes, and certain proteins, even in the absence of phospholipid. Elevated levels of aPL are common in the antiphospholipid syndrome (APS) and are often associated with the development of pathogenic clinical features, such as arterial and venous thromboembolic events, recurrent fetal loss and thrombocytopenia.
The epitopes for aPL have been a subject of controversy. It has been proposed that, as a result of noncovalent protein-lipid interactions, novel conformational epitopes are formed on the plated CL, on ß2GP1, or on an admixture of these 2, or that ß2GP1 alone is the antigen.1–4 Cardiolipin is a phospholipid (PL) that contains 4 unsaturated fatty acid chains, and we have previously demonstrated that CL is rapidly oxidized when plated on microtiter wells and exposed to air, as it is done in conventional solid-phase anti-cardiolipin (aCL) immunoassays.5 We have also shown that human aCL antibodies from APS patients bind only to oxidized cardiolipin (oxCL) and/or to oxCL-ß2-glycoprotein 1 (ß2GP1) adducts but not to a "reduced" cardiolipin that is unable to undergo oxidation.6,7 We have proposed that some, if not most, aCL bind to neo-epitopes generated when CL undergoes oxidation. The formation of these neo-epitopes between oxPL and associated proteins would be analogous to low-density lipoprotein (LDL) oxidation, in which oxidized phospholipids covalently bind to apoB generating similar "oxidation-specific" immunogenic neo-epitopes.8,9
During oxidative modification of LDL, there is an initiation of peroxidation of the polyunsaturated fatty acids present in surface PL. This leads to the formation of a variety of highly reactive breakdown products, which are immunogenic.10 The recent demonstration that circulating LDL contains significant amounts of CL11 (previously thought to be only intracellular) suggests that CL may also be susceptible to oxidation during LDL oxidation, and that many aCL and anti-oxLDL antibodies may be directed at similar epitopes. In fact, aCL from patients with systemic lupus erythematosus are known to cross-react with oxidized LDL.6,12 We reasoned that any PL containing an unsaturated fatty acid, eg, CL in LDL, would be susceptible to oxidation by mechanisms analogous to those causing LDL oxidation, and could thus potentially generate immunogenic neo-epitopes. Here we have further tested this hypothesis by demonstrating the presence of autoantibodies to oxCL in LDL receptor-deficient mice (LDLR–/–) that have not been exogenously immunized. LDLR–/– mice fed a high-fat diet are exposed to oxidative stress and generate high autoantibody titers to various epitopes on oxLDL and develop atherosclerosis.13 We describe the cloning and characterization of a natural monoclonal IgM anticardiolipin antibody (LRO1) from a nonimmunized LDLR–/– mouse, and show that it not only binds to oxCL but also binds to oxLDL, apoptotic cells, and atherosclerotic lesions. These observations provide new insights into mechanisms involved in the generation of aCL.
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Methods |
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Human Subjects
LRO1 epitopes on LDL particles were measured from plasma samples of 113 human subjects. All but 1 of the subjects had rheumatoid arthritis, according to American Rheumatism Association 1987 criteria. All patients were recruited from the Oulu University Hospital, Finland, and gave informed consent. The study was approved by the Ethical Committee of the Oulu University Hospital, and followed the Declaration of Helsinki.
Cloning of Monoclonal Antibody LRO1
Splenocytes from an 8-month-old LDLR–/– mouse on a high-fat diets for 6 months were fused with P3x63Ag8.653.1 myeloma cell line using standard methods. The mouse was not exogenously immunized prior to the fusion (a naive LDLR–/– mouse). Hybridoma supernatants were screened for a variety of oxidation-specific epitopes, including oxCL, using the chemiluminescence immunoassay as described (please see online Methods at http://atvb. ahajournals.org). After fusion, the hybridoma with highest binding to oxCL (LRO1) was selected for further characterization and cloning by limiting dilution (3 rounds). LRO1 was isotyped as IgM and purified using IgM HiTrap and superose 6HR 10/30 columns (Pharmacia Biotech). The LDLR–/– mice in C57BL/6 background were bred in-house in Dr Witztum’s laboratory.
Antigen Preparation and Chemiluminescent Immunoassay
Freshly isolated human LDL was Cu2+-oxidized (Cuox-LDL) as previously described.9 Liposomes of oxCL, reduced cardiolipin (CLred), phosphatidylcholine (PC), lyso-PC, phosphatidylethanolamine (PE) (Avanti Polar Lipids, Alabaster, Ala) and oleic acid (Sigma-Aldrich) were generated by sonicating lipids (1 mg/mL) in phosphate-buffered saline for 5 minutes. Human ß2GP1was purchased from HTI Bio-Products (Ramona, Calif). The chemiluminescent immunoassays were performed as described in detail in the supplement (please see online Methods at http://atvb.ahajournals.org).
RNA Isolation, cDNA Synthesis, and Sequence Analysis of VH and V
Please see online Methods at http://atvb.ahajournals.org.
Immunohistological Staining of Atherosclerotic Lesions
Paraformaldehyde-fixed, paraffin-embedded sections of human brain arteries and an aortic arch of Watanabe heritable hyperlipidemic rabbits were immunostained as described in online Methods at http://atvb.ahajournals.org.
Flow Cytometry Analysis and Immunofluorescence Microscopy for LRO1 Binding to Apoptotic Cells
Apoptosis was induced in human Jurkat T-cells by UV-irradiation with 20 mJ/cm2 (FB-UVXL-1000 UV Crosslinker; Fisher Biotech), followed by overnight incubation at 37°C. The cells were incubated with LRO1 (3 to 5 µg/mL) or control mouse IgM antibody (10 µg/mL) for 60 minutes at 4°C, followed by a wash and incubation with 5 µg/mL of fluorescein-conjugated (fluorescein isothiocyanate [FITC]) anti-mouse IgM secondary antibody (BD Pharmingen) for 35 minutes at 4°C. For flow cytometry analysis, cells were incubated with 1 µg/mL of propidium iodide (PI) for 10 minutes and immediately analyzed by a FACScan instrument (Becton Dickinson). Data were analyzed using FCS Express software.
For immunofluorescence microscopy studies, the cells were first incubated with MitoTracker (Molecular Probes) and then fixed and permeabilized with 3.7% paraformaldehyde containing 0.8% saponin for 20 minutes at 4°C. Incubations with primary and secondary antibodies (as noted) were followed by staining with 1 µg/mL of Hoechst dye (Sigma-Aldrich), and the cells were spun down on glass slides using cytospin (ThermoShandon). Images were captured using a DeltaVision deconvolution microscopic system operated by SoftWorx software(Applied Precision) as described.14
Measurement of LRO1 Epitopes in Human Plasma LDL and IgG aCL Titers
The content of LRO1 epitopes in LDL from human plasma samples was measured with a chemiluminescent capture assay as previously described in detail.15 Human plasma (1:25 dilution) was added to anti-apoB-100 antibody (5 µg/mL) (Biodesign) coated wells. LRO1 epitopes were determined on the captured apoB-100 particles with biotinylated LRO1 using alkaline-phosphatase-labeled avidin. In parallel wells, the amount of apoB-100 captured was measured using biotinylated anti-apoB-100 antibody. The results are expressed as a ratio of LRO1 binding divided by anti-apoB-100 binding. For aCL IgG measurement, please see online Methods at http://atvb.ahajournals.org.
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Results |
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LRO1 Binding to oxCL, CLred, and ß2GP1
We tested LRO1 binding to oxCL and CLred (a reduced cardiolipin analogue containing only saturated fatty acids that is unable to undergo oxidation). Figure 1A shows that LRO1 had high binding only to oxCL, and did not bind unoxidized CLred. We also tested LRO1 binding to a possible cofactor ß2GP1, with and without the presence of oxCL. LRO1 did not bind to purified human ß2GP1, a reported cofactor for some aCL, nor did ß2GP1 enhance the binding of LRO1 to oxCL (Figure 1A). The specificity of LRO1 binding to oxCL was further demonstrated in a competition immunoassay using liposomes containing oxCL, CLred, phosphatidylcholine (PC) or phosphatidylethanolamine (PE) as competitors (Figure 1B). LRO1 binding to plated oxCL was only competed by liposomes containing oxCL but not with liposomes containing CLred, PC, or PE (Figure 1B). LRO1 did not bind to liposomes containing lyso-PC, PS, or oleic acid (data not shown).
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LRO1 Binding to oxLDL
To further evaluate whether LRO1 recognized epitopes in LDL after it undergoes oxidation, we studied the binding of LRO1 to LDL oxidized by exposure to copper ions (oxLDL). Figure 2A demonstrates that LRO1 exhibited dose-dependent binding to oxLDL, but did not bind to native-LDL. Furthermore, the binding of LRO1 to oxLDL was specifically competed by oxLDL and by LDL to which oxCL had been added, but not by native LDL (Figure 2B). The binding of LRO1 to oxLDL, but not to native LDL demonstrates that cardiolipin present in native LDL is also oxidized when LDL is oxidized, generating neo-epitopes recognized by LRO1.
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V Gene Family Usage and V-(D)-J Junction Analysis of VH and V of LRO1
To study the genetic origin of LRO1 we sequenced the immunoglobulin variable regions of its heavy and light chains. The LRO1-VH originates from VH9 germline gene (VHGam3.8) with only one nucleotide difference leading to one amino acid change in framework region one (FR1) (Table I, please see http://atvb.ahajournals.org). The complementary-determining region 3 of the heavy chain (CDR-H3) includes nucleotide insertions both in V-D junction (1 palindromic and 2 nucleotides) and D-J junction (6 nucleotides) (Table I). The VH D region (D-FL16.1*01) is nonmutated and the J region (JH1*03) has 1 nucleotide change (no amino acid change) compared with the germlines (Table II). The V sequence of LRO1 is 100% identical to V19–20 germline (Table I) and J sequence is 100% identical to J1 and there is 1 additional palindromic nucleotide in the V-J junction (Table II). This suggests that LRO1 is a natural antibody with high homology to mouse germline antibody sequences.
LRO1 Binding to Atherosclerotic Lesions
To demonstrate the existence of epitopes recognized by LRO1 in vivo, sections containing atherosclerotic lesions from human brain arteries (Figure 3A) and rabbits aortas (Figure 3B and 3C) were stained with LRO1. LRO1 immunostained atherosclerotic lesions, and the LRO1 binding seemed to be localized more in the necrotic core of the lesions.
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LRO1 Binding to Apoptotic Cells
We hypothesized that LRO1 epitopes in atherosclerotic lesions may originate from the oxLDL in the lesion.16 However, another likely etiology of oxCL could be the presence of apoptotic cells, which are know to accumulate in atherosclerotic lesions, especially in the necrotic area. Apoptotic cells have recently been shown to contain oxidized cardiolipin leaking from the mitochondria.17 To test this hypothesis, we investigated the binding of LRO1 to apoptotic Jurkat cells using flow cytometry. LRO1 did not bind to normal viable cells (Figure 4F) with low PI staining (Figure 4E), but displayed increased binding to nonpermeabilized apoptotic Jurkat cells (Figure 4B) that had high PI staining (late apoptotic cells, Figure 4A). The binding of LRO1 to late apoptotic cells was substantially increased when the cells were permeablized with saponin (Figure 4D), suggesting that many LRO1 epitopes were intracellular. LRO1 did not bind to permeabilized viable cells (data not shown). The flow cytometry analysis was further confirmed by immunofluorescence microscopy, where LRO1 bound to permeabilized late apoptotic cells (Figure 5A). No binding was observed with a control mouse IgM antibody to the same cells (Figure 5B). Using deconvolution microscopy, we demonstrated intense intracellular LRO1 binding (FITC, green color) inside a cell that had undergone apoptosis (fragmented dense nucleus visualized with blue Hoechst dye) and not to a cell that had intact nucleus (Figure 5C). Costaining with Mitotracker (red color), which stains active mitochondria, indicate a depletion of intact mitochondria in the apoptotic cell with the generation of enhanced oxidative conditions and oxCL. This supports the hypothesis that oxCL from dying cells could be an antigen leading to expansion of antibodies such as LRO1.
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LRO1 Epitopes on Human LDL
Circulating LDL has been shown to contain epitopes of oxidized PC-containing phospholipids.18 Because human LDL has been shown to contain cardiolipin,11 we investigated if LRO1 epitopes were also present in vivo in plasma LDL of 113 subjects with rheumatoid arthritis in which aCL antibodies are known to be increased. In this enzyme-linked immunosorbent assay, apoB-100 containing lipoproteins were captured from human plasma using an anti-apoB-100 antibody. The amount of LRO1 epitopes on captured apoB-100 particles was then measured with biotinylated LRO1.15 These data indicated that there was up to a 10-fold variation in the amount of oxCL present in plasma LDL measured with LRO1 antibody among the subjects (Figure 6). We also measured the levels of IgG aCL antibody titers to oxCL in these subjects to assess if there was a relation between the amount of LRO1 epitopes in LDL and the antibody titers to oxCL. In fact, there were varying levels IgG antibody titers to oxCL and these were positively related to the oxCL in apoB-100 particles measured with LRO1 antibody (r=0.32, P=0.0004) (Figure 6). We also measured sensitive CRP levels of these plasma samples to study if the amount of LRO1 epitopes in plasma LDL were related with the severity of the inflammatory disorder. There was no correlation between sensitive CRP levels and LRO1 epitopes in plasma LDL (data not shown).
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Discussion |
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We have previously proposed that some aCL bind to neo-epitopes of oxidized phospholipids or to neo-epitopes generated by adduct formation between reactive breakdown products of oxidized phospholipids and associated proteins.5–7,19 We have also suggested that oxCL belongs to a novel class of pathogen-associated molecular patterns (PAMPs).10 To provide evidence in support of these hypothesis, we cloned a natural antibody to oxCL from a nonimmunized LDLR–/– mouse, and showed that it bound specifically to oxCL and oxLDL, but not to unoxidized, reduced cardiolipin or native LDL. More importantly, the VH andV
sequence analysis revealed that LRO1 was highly homologous to germline genes, and, therefore, is a natural antibody. We demonstrated that epitopes for LRO1 are present in vivo in human atheromas, on apoptotic cells and in human plasma LDL, suggesting that these may share common idiotypic determinants responsible for induction of LRO1. These data strongly suggests that CL oxidation is needed for its immunogenicity, and that oxCL is indeed a PAMP of innate immunity.
It is widely accepted that lipoprotein oxidation and formation of oxLDL plays an important role in atherogenesis. During LDL oxidation, a large number of oxidative neo-epitopes are formed as a result of oxidative decomposition of the lipids of LDL and adduct formation between lipid peroxidation products and reactive amine groups of apolipoproteins.10 Decomposition of the oxidized fatty acids generates a wide spectrum of reactive molecular species, such as malondialdehyde and 4-hydroxynonenal,20 as well as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine.21 These reactive aldehydes can further modify autologous molecules, including both the protein and the lipid moiety of LDL. Theoretically, hundreds of such different modified structures can occur, and we have named these epitopes "oxidation-specific epitopes." We have hypothesized that these oxidation-specific epitopes are a class of PAMP that are recognized by natural antibodies and other innate immunity receptors.10 Various oxidation-specific epitopes induce strong humoral immune responses, and circulating autoantibodies to several oxidation-specific epitopes have been demonstrated in humans and animal models of atherosclerosis.13,22,23 Of particular interest to the present studies, apoE–/– mice have previously been shown to have expansion of IgG and IgM antibodies to oxCL.23 More importantly, the autoantibody titers to oxCL in these mice were related to the extent of atherosclerosis and aortic levels of isoprostane F2
-VI, which is a specific and sensitive quantitative marker of in vivo lipid peroxidation.23 Considering that cardiolipin is also one of the phospholipids present in lipoproteins,11 our data strongly suggest that LDL oxidation also induces immune response to oxCL epitopes. In fact, LRO1 immunoreactivity in plasm was only found in apoB-100 containing lipoproteins. In a set of 15 plasma samples, we captured high-density lipoprotein particles with an anti-apoAI antibody and measured LRO1 epitopes using a method similar to the measurement of LRO1 epitopes in LDL apoB-100–containing particles. Almost no LRO1 immunoreactivity was found in the high-density lipoprotein particles (data not shown). Thus, in addition to the previously well known and extensively studied immunoreactivity of other LDL oxidation products (eg, those mentioned), cardiolipin can also undergo oxidation and lead to the expansion of clones producing autoantibodies binding to it.
Natural antibodies are often defined as antibodies that appear in normal individuals in the complete absence of any exogenous antigenic stimulation and have an important role in providing a first line of defense against invading pathogens.24 In addition, natural antibodies are also thought to have dual specificities, not only binding to pathogens but also they are believed to play a "housekeeping" role by recognizing and removing self-antigens such as senescent cells, cell debris, and apoptotic cells.24 Natural antibodies demonstrate a remarkably conserved repertoire that includes a broad specificity for self-antigens, and we have previously suggested that many oxidation-specific epitopes are a class of common idiotypic determinants, i.e. PAMPs, which are present in oxLDL, apoptotic cells and some infectious agents.10
We have shown earlier that one such PAMP is the PC head group of oxidized phosphatidylcholine, which shows immunologic identity to the PC present on the cell wall of many bacteria, such as streptococcus pneumoniae.25 In addition, we demonstrated that expansion of natural antibodies to PC confers atheroprotection.25 Cardiolipin is a negatively charged anionic phospholipid originally found in membranes of bacteria26 and inner mitochondrial membrane.27 However, it was recently reported to be present also in human plasma, more than 94% of it located in lipoproteins, with a majority (67%) located in the LDL density fraction.11 Because of the high level of unsaturated fatty acid chains (mainly linoleic acid) in CL, it is extremely susceptible to oxidation, and we have previously demonstrated that CL oxidation is needed to generate epitopes for many aCL antibodies.5–7 This was also true for LRO1. Both heavy and light chains of LRO1 were highly homologous to the germline genes, which makes it a natural antibody. The sequence analysis of LRO1 VH revealed that this monoclonal antibody is derived from the small VH9 (VGAM3.8) family, which has been found in monoclonal antibodies of diverse specificities, but the overall representation is considered to be low.28–31 Of special interest is that there is very little, if any, polymorphism in the germline IgH haplotypes of VGAM3.8 sequences between different mouse strains (C57BL/6, BALB/c and NZW strains), further supporting the hypothesis that LRO1 is highly conserved and that it may have been selected and expanded in response to common idiotypic determinants or pathogens,31 such as, eg, oxCL, found in apoptotic cells, but also in membranes of bacteria under inflammatory settings.26 Thus, our data suggest that oxCL is another PAMP that is present in oxLDL, apoptotic cells and in certain bacteria.
oxLDL is known to be present in atherosclerotic lesions, and we showed that LRO1 also recognizes epitopes in lesions. However, the origin of LRO1 epitopes in the lesions could also be derived from oxCL in apoptotic cells, which are known to accumulate in the lesions.16 In cells undergoing apoptosis CL oxidation is an early event involved in the formation of the mitochondrial permeability transition pore that facilitates release of cytochrome c (along with several other pro-apoptotic factors) from the mitochondria into the cytosol.17,32 Although apoptotic cells are normally considered to be cleared by phagocytes without stimulating inflammatory or immune response, there may be, under certain conditions, pathological accumulation of increased numbers of apoptotic cells which may induce immune responses.16,33–36 It has been postulated as a potential mechanism responsible for some autoimmune diseases.33,36 Indeed, we demonstrated that LRO1 bound to apoptotic cells, supporting our previous studies that many aPL to oxidized lipid protein adducts bind to common antigenic determinants generated in cells during apoptosis.36 Thus, we provide data suggesting that the "immunogenic trigger" for some aCL binding to oxCL may originate from oxCL present in LDL and/or in apoptotic cells, which are both components of atherosclerotic lesions. In addition, apoptotic cells are known to accumulate in models of lupus erythematosus, a condition in which high titers of aCL are found.37 This also suggests a role for natural antibodies in the recognition and removal of senescent cells, cell debris, and other neo-self epitopes, and that their primary role is actually to protect from autoimmunity. It can be hypothesized that the physiological "housekeeping" role of natural antibodies to recognize altered self may become especially important under conditions that lead to increased production of stress-induced self-antigens, such as occurs in atherosclerosis or in similar inflammatory conditions.
In summary, we have demonstrated by cloning a natural monoclonal antibody to oxCL that oxCL present in oxLDL, apoptotic cells, and atherosclerotic lesions is an important antigenic determinant. The knowledge that some aCL antibodies, such as LRO1, are natural antibodies and oxidation-dependent may give insight into the pathogenic events underlying the clinical manifestations of diseases with increased titers of aCL antibodies, such as lupus or atherosclerosis.
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Acknowledgments |
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Sources of Funding
This work was supported by the Research Council for Health of the Academy of Finland (Y.A.K), the Finnish Foundation for Cardiovascular Research (S.H.), and Sigrid Juselius Foundation (S.H.), and grants from the NHLBI HL57505, HL56989 (S.H. and J.L.W), and HL081862 (Y.I.M.), and American Heart Association grant 0530159N (Y.I.M.).
Disclosures
None.
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Footnotes |
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Original received January 25, 2006; final version accepted June 7, 2006.
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