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Originally published In Press as doi:10.1194/jlr.M600414-JLR200 on January 29, 2007

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Journal of Lipid Research, Vol. 48, 768-781, April 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

The association of C-reactive protein with an oxidative metabolite of LDL and its implication in atherosclerosis

Masako Tabuchi*, Katsumi Inoue{dagger},§, Hitomi Usui-Kataoka**, Kazuko Kobayashi*, Misako Teramoto{dagger}, Koji Takasugi**, Kenichi Shikata**, Masahiro Yamamura**, Kenji Ando{dagger}{dagger}, Keiichiro Nishida§§, Junko Kasahara***, Noriaki Kume{dagger}{dagger}{dagger}, Luis R. Lopez§§§, Kazuaki Mitsudo§, Masakiyo Nobuyoshi{dagger}{dagger}, Tatsuji Yasuda*, Toru Kita{dagger}{dagger}{dagger}, Hirofumi Makino** and Eiji Matsuura1,*

* Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan
** Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan
§§ Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan
{dagger} Department of Pathology, Kokura Memorial Hospital, Kitakyushu 802-8555, Japan
{dagger}{dagger} Department of Cardiology, Kokura Memorial Hospital, Kitakyushu 802-8555, Japan
§ Department of Cardiovascular Medicine, Kurashiki Central Hospital, Kurashiki 710-8602, Japan
*** Department of Internal Medicine, Okayama Central Hospital, Okayama 700-0017, Japan
{dagger}{dagger}{dagger} Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan
§§§ Corgenix, Inc., Broomfield, CO 80020

Published, JLR Papers in Press, January 29, 2007.

1 To whom correspondence should be addressed. e-mail: eijimatu{at}md.okayama-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
C-reactive protein (CRP) is one of the strongest independent predictors of cardiovascular disease. We have previously reported that oxidized LDL (oxLDL) interacts with ß2-glycoprotein I (ß2GPI), implicating oxLDL/ß2GPI complexes as putative autoantigens in autoimmune-mediated atherosclerotic vascular disease. In this study, we investigated the interaction of CRP with oxLDL/ß2GPI complexes and its association with atherosclerosis in patients with diabetes mellitus (DM). CRP/oxLDL/ß2GPI complexes were predominantly found in sera of DM patients with atherosclerosis. In contrast, noncomplexed CRP isoforms were present in sera of patients with acute/chronic inflammation, i.e., various pyrogenic diseases, rheumatoid arthritis (RA), and DM. Immunohistochemistry staining colocalized CRP and ß2GPI together with oxLDL in carotid artery plaques but not in synovial tissue from RA patients, strongly suggesting that complex formation occurs during the development of atherosclerosis. Serum levels of CRP correlated with soluble forms of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and oxLDL/ß2GPI complexes correlated with total cholesterol and hemoglobin A1c. Thus, the generation of CRP/oxLDL/ß2GPI complexes seems to be associated with arterial inflammation, hyperglycemia, and hypercholesterolemia. CRP/oxLDL/ß2GPI complexes can be distinguished from pyrogenic noncomplexed CRP isoforms and may represent a more specific and predictive marker for atherosclerosis.

Supplementary key words ß2-glycoprotein I • oxidized LDL/ß2-glycoprotein I complexes • diabetes mellitus • oxidized LDL

Abbreviations: APS, antiphospholipid syndrome; AT, atherosclerosis; CRP, C-reactive protein; CVD, cardiovascular disease; DM, diabetes mellitus; ß2GPI, ß2-glycoprotein I; HbA1c, hemoglobin A1c; hsCRP, high-sensitivity CRP; ICAM-1, intercellular adhesion molecule-1; IMT, intima media thickness; MAb, monoclonal antibody; oxLDL, oxidized LDL; pI, isoelectric point; RA, rheumatoid arthritis; ROC, receiver operating characteristic; T-chol, total cholesterol; VCAM-1, vascular cell adhesion molecule-1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atherosclerosis is a pathological condition in which arteries undergo gradual intima thickening, causing decreased elasticity, narrowing, and reduced blood supply. The atherosclerotic involvement of these blood vessels brings about the distinct clinical manifestations of cardiovascular disease (CVD). A characteristic histopathologic finding of atherosclerosis is the focal appearance of macrophage-derived lipid-laden foam cells. The cholesterol that accumulates in foam cells is derived from circulating lipids, mainly from oxidized LDL (oxLDL) (1, 2) generated by vascular inflammation and oxidative stress from different types of pathologic injury. OxLDL is also a proinflammatory chemoattractant agent for macrophages and T lymphocytes, cytotoxic for endothelial cells, and stimulates the release of soluble inflammatory molecules. All of these events not only help to perpetuate a cycle of vascular inflammation and lipid dysregulation within the arterial walls but also create an endothelial prothrombotic state that complicates late stages of atherosclerosis (i.e., arterial thrombosis).

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of a heterogenous group of antiphospholipid antibodies (Abs) with arterial and/or venous thromboembolic complications, and/or pregnancy morbidity (3, 4). Antiphospholipid Abs are directed against epitopes that include proteins such as ß2-glycoprotein I (ß2GPI) (4–6), lipids, and protein-lipid complexes. We have demonstrated that anti-ß2GPI auto-Abs also recognize oxLDL/ß2GPI complexes (4, 7–10). Further, the in vitro macrophage uptake of oxLDL/ß2GPI complexes was significantly increased by IgG anti-ß2GPI Abs. (4, 7–10) Thus, findings suggesting that IgG anti-ß2GPI auto-Abs may be proatherogenic renewed interest in the immunologic mechanisms participating in atherogenesis (4, 11, 12).

ß2GPI interacts with oxLDL via oxidatively induced ß2GPI-specific ligands, e.g., 7-ketocholesterol having an {omega}-carboxyl acyl chain. This interaction may take place in the arterial intima of atherosclerotic lesions, producing stable and nondissociable oxLDL/ß2GPI complexes (4, 8–10). These complexes would then be released into the circulation. In addition to systemic autoimmune diseases (15, 16), oxLDL/ß2GPI complexes have been found in nonautoimmune chronic inflammatory diseases that develop atherosclerosis, such as diabetes mellitus (DM) (13) and chronic nephritis (14). OxLDL/ß2GPI complexes may represent a serologically relevant form of circulating oxLDL, because oxLDL injected intravenously into experimental animals was immediately removed from circulation by the liver (17).

C-reactive protein (CRP) is an acute-phase reactant that belongs to the highly conserved "pentraxin" family and plays a major role in innate immunity (18–20). CRP binds to a range of autologous and exogenous ligands, including phosphorylcholine originating from either the capsular polysaccharide component of microorganisms, native LDL, oxLDL, or apoptotic cells. This interaction moderates the clearance of opsonized CRP by macrophages (21–24). Further, CRP binds to multiple ligands, activating the classical complement pathway, which in turn enhances phagocytosis via complement receptors. Epidemiologic studies have shown an association between CRP and the risk for CVD (25, 26). Multiple clinical studies have demonstrated a predictive relationship between increased CRP levels and atherothrombotic events. It has also been suggested that CRP is not only a serologic marker for atherosclerosis but also a causal risk factor, stimulating adhesion molecule expression and chemokine production by endothelial cells (27, 28). Thus, CRP aids in the first line of host defense against infection and is also involved in inflammatory processes that promote atherosclerosis (atherothrombosis) (29).

In the present study, circulating CRP/oxLDL/ß2GPI complexes demonstrated by ELISA and immunoblot analysis were predominantly detected in DM patients with atherosclerosis. Because CRP/oxLDL/ß2GPI complexes were not detected in patients with pyrogenic diseases and rheumatoid arthritis (RA), they may be a part of the CVD predictor, i.e., high-sensitivity CRP (hsCRP). Immunohistochemistry also revealed the possibility that the complexes are formed in atherosclerotic plaques and released into the circulation. Thus, CRP/oxLDL/ß2GPI complexes, which can be distinguished from pyrogenic noncomplexed CRP isoforms, may be a more specific and predictive marker for atherosclerosis (atherothrombosis).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and specimens
The present cross-sectional study was performed at Okayama University Hospital (Okayama, Japan). The protocols were approved by the Institutional Ethical Review Board of Okayama University Hospital. Informed consent was given by all participants.

Type 2 DM patients (n = 125) fulfilling the diagnostic criteria of the World Health Organization (WHO) (30) were enrolled in the present study. The diagnosis of DM was based on the presence of chronic hyperglycemia and metabolic disturbances of lipid, carbohydrate, and protein metabolism due to defects in insulin production or activity. Impaired fasting glycemia and/or glucose tolerance test results were used according to WHO established criteria. DM patients with significant CVD events, pregnancy, inflammatory disease, and/or steroid use were excluded. DM patients had a male to female ratio of 73:52 and a mean age of 61.4 ± 13.3 years (mean ± SD; range 34–67 years). Age-matched RA patients (n = 48) and healthy subjects (n = 48) were enrolled. All RA patients met the American College of Rheumatology revised criteria (31). RA patients with DM and/or significant CVD events were excluded. RA patients had a male to female ratio of 7:41 and a mean age of 64.0 ± 10.0 years (range 33–80 years). Thirty-five patients with acute pyrogenic diseases (i.e., acute enteritis, acute pancreatitis, acute prostatitis, acute pyelonephritis, acute tonsillitis, appendicitis, bronchitis, diverticulitis of colon, cystitis, epididymitis, femoral necrotizing fasciitis, gonitis, fever of unknown cause, infectious enteritis, mesenteric panniculitis, pharyngitis, or pneumonia) without DM, CVD, or collagen diseases were enrolled. All serum samples were stored at –80°C until use.

Tissue samples were prepared from human carotid endarterectomy specimens from 17 patients with transient ischemic attack or minor completed strokes before surgery. Synovial tissue samples were prepared from five RA and five osteoarthritis patients. All tissues were fixed with neutral buffered formalin, embedded in paraffin, and sectioned by standard procedures.

Abs and reagents
Monoclonal antibody (MAb) WB-CAL-1 (IgG2a, {kappa}) was derived from nonimmunized NZW x BXSB F1 male mice, an animal model of APS (32). WB-CAL-1 MAb binds to ß2GPI complexed with oxLDL or lipid vesicles containing anionic phospholipids or oxidized cholesteryl esters derived from oxLDL, but not to the noncomplexed form of the protein (5, 7–10). In contrast, another anti-ß2GPI MAb, Cof-23 (IgG1, {kappa}), obtained from BALB/c mice immunized with human ß2GPI, recognizes both the noncomplexed and lipid-complex ß2GPI structures (33). Two anti-apolipoprotein B-100 (apoB-100) MAbs were established from BALB/c mice immunized with human native LDL and Cu2+-oxLDL, respectively: N2E10 (IgG2a, {kappa}), which recognizes apoB-100 present in either native LDL or oxLDL; and O1F9 (IgG2a, {lambda}), which recognizes apoB-100 present in oxLDL but not in native LDL (i.e., anti-oxLDL Ab). A MAb against scavenger receptor SR-PSOX (34) was used for immunohistochemistry staining. Commercially available goat anti-CRP Abs were obtained from Bethyl Laboratories, Inc. (Montgomery, TX); mouse anti-CD68 MAb and mouse anti-{alpha} actin of smooth muscle cells (SMC{alpha}) MAb from Dako (Kyoto, Japan); horseradish peroxidase (HRP)-labeled rabbit anti-CRP Abs from Medical and Biological Laboratories (Nagoya, Japan); and human CRP, purified from pleural fluid (Chemicon International, Temecula, CA) and from serum (Calbiochem, EMD Biosciences, Inc., La Jolla, CA). Purity of these CRP preparations was checked by SDS-PAGE, yielding a 24 kDa single band under reducing or nonreducing conditions. Both CRP preparations were extensively dialyzed against 10 mM Tris, 150 mM NaCl, 1.25 mM CaCl2, pH 7.4 (Tris buffer) to remove NaN3 before use. ß2GPI was purified from healthy human plasmas, as described (10). Other chemicals were obtained from commercial sources and were of reagent-grade quality.

Isolation and oxidation of LDL
LDL (1.019<d<1.063) was isolated by ultracentrifugation from fresh healthy human plasma, as described (10). LDL (100 µg/ml of apoB equivalent) in 10 mM Hepes and 150 mM NaCl, pH 7.4, (Hepes buffer) was oxidized by incubation with 5 µM CuSO4 for 12 h at 37°C. Aliquots were taken to determine thiobarbituric acid-reactive substances and electrophoretic migration in agarose gels.

Reduction of endogenous and/or exogenous endotoxin (lipopolysaccharide) from reagents
Affinity chromatography using a Detoxi-Gel column (Pierce; Rockford, IL) was performed to remove exogenous endotoxin that might have contaminated the CRP and ß2GPI preparations during isolation. In experiments to assess endotoxin involvement in complex formation, additional purification steps were performed. For example, endotoxin-free water and sterile materials, including glassware, which were either commercially apyrogenic or depyrogenated by steam autoclaving and/or dry heat, were used during LDL isolation. Endotoxin levels were determined by the Limulus amoebocyte lysate assay.

ELISA for oxLDL complexes
ELISA for oxLDL/ß2GPI complexes This procedure was performed as previously described (10). Briefly, anti-ß2GPI MAb, WB-CAL-1, was adsorbed onto microtiter plates (Immulon 2HB; Thermo Labsystems, Franklin, MA) by incubating at 8 µg/ml (dissolved in Hepes buffer, 50 µl/well) at 4°C overnight. After blocking with Hepes buffer containing 1% skim milk, samples diluted 1:100 with Hepes buffer containing 0.5% skim milk were added to the wells (100 µl/well) to be incubated for 2 h. The wells were then incubated with HRP-labeled anti-human apoB-100 MAb (N2E10). Extensive washing between steps was performed with Hepes buffer containing 0.05% Tween 20. Color was developed with tetramethylbendizine and H2O2. The reaction was terminated, and optical density at 450 nm was measured.

ELISA for CRP/oxLDL complexes Captured anti-apoB-100 MAb, N2E10, was adsorbed onto microtiter plates (Immulon 2HB; Thermo Labsystems) by incubating at 8 µg/ml (dissolved in Hepes buffer, 50 µl/well) at 4°C overnight. After blocking with 10 mM Tris, 150 mM NaCl, 1.25 mM CaCl2, pH7.4, (Tris buffer) containing 0.5% BSA, samples diluted 1:100 with Tris buffer containing 0.2% BSA were added to each well and incubated for 2 h. The wells were subsequently incubated with HRP-labeled anti-CRP Abs for 1 h. Extensive washing between steps was performed with Tris buffer containing 0.05% Tween 20. Further steps were performed as described above for oxLDL/ß2GPI complexes.

ELISA for CRP/oxLDL/ß2GPI complexes The assay was similar to that for the ELISA for CRP/oxLDL complexes, with the exception that captured N2E10 MAb was replaced by anti-ß2GPI MAb WB-CAL-1 on the plate.

Nondenaturing PAGE (native PAGE) and immunoblot analysis
Nondenaturing PAGE (native PAGE without SDS) was carried out on 2–15% polyacrylamide gradient gels according to the method of Krauss and Burke (35), with slight modifications. A pH of 8.3 was used for running the gels to facilitate the characterization of LDL subclasses. To avoid the influence of protein charge, protein markers having similar isoelectric points (pIs) are generally used in nondenaturing PAGE. A set of commercially available molecular mass markers (Daiichi Pure Chemicals; Tokyo, Japan) for nondenaturing PAGE composed of thyroblobulin (669 kDa, pI 4.6), ferritin (443 kDa, pI 4.5), lactate dehydrogenase (140 kDa, pI 4.0), BSA (66 kDa, pI 4.7), and soybean trypsin inhibitor (20.1 kDa, pI 4.50) was used for the study. Then the proteins were transferred to a polyvinylidene difluoride membrane, and immunoblot was performed with anti-CRP Abs anti-ß2GPI MAb (Cof-23) or anti-apo-B100 MAb (N2E10).

Immunohistochemistry
Details of the immunohistochemical staining procedure were previously described (36). Pretreatment of human carotid endarterectomy and synovial tissue sections in an oil bath (97°C, 20 min in 0.1 M Tris-HCl, pH 6.0) was performed after deparaffinization according to the modified method of Shi, Key, and Kalra (37), except for the CRP staying run. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 in methanol for 30 min, followed by blocking with 2% normal goat serum. Immunohistochemical staining of adjacent sections was carried out using Abs against CD68, SMC{alpha}, CRP, SR-PSOX, and apoB in either native LDL or oxLDL (N2E10), apoB in oxLDL (O1F9), noncomplexed and complexed forms of ß2GPI (Cof-23), and complexed form of ß2GPI (WB-CAL-1).

Other clinical and biological parameters
Intima media thickness (IMT) was measured by B-mode ultrasonography. Three IMT measurements of diastolic images on each side at 10 mm before or after the carotid bifurcation were obtained. Mean IMT was calculated for each point, and the highest value (maximum IMT) was recorded for each subject and was defined as the distance from the lumen-intima interface to the intima-adventitia interface. An IMT cutoff value of 0.75 mm was used to diagnose atherosclerosis. Serum hsCRP was measured by nephelometry, a latex particle-enhanced immunoassay (N-Latex CRPII; Dade Behring, Tokyo, Japan). Total cholesterol (T-chol), hemoglobin A1c (HbA1c), and CRP were measured by routine laboratory methods. Soluble forms of intracellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) were measured by commercially available ELISAs (R and D Systems, Inc.; Minneapolis, MN).

Statistical analyses
Statistical analysis was performed by StatView software (Abacus Concepts; Berkeley, CA). The Student t-test was used to assess possible correlation between levels of CRP or three types of oxLDL complexes and the occurrence of disease. The correlation between two variables was evaluated by the Pearson correlation test. Fisher's exact test was used to compare the appearance of biological markers, CRP and oxLDL complexes. Ninty-five percent confidence interval (95% CI) was calculated by Woolf's method. The level of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Formation of CRP/oxLDL/ß2GPI complexes
The nature of the interaction between oxLDL and ß2GPI has been previously characterized and described (8–10). We first investigated the interaction among Cu2+-oxLDL, ß2GPI, and CRP. Figure 1A –C depicts the calcium dependency of the interaction of oxLDL with CRP and oxLDL with ß2GPI. Figure 1A shows that the interaction between oxLDL and ß2GPI was calcium independent from 0 to 5 mM of Ca2+ ion. In contrast, the interaction between oxLDL and CRP was calcium dependent (Fig. 1B). The complexes were not dissociated even if excess EDTA or Ca2+ ion was added after the completion of the reaction (data not shown). At physiological calcium concentrations (approximately 1.25 mM), both the calcium-independent and -dependent interactions took place (Fig. 1A–C). CRP/oxLDL/ß2GPI complexes were formed proportionally when these three components were coincubated (Fig. 1C). No direct interaction between CRP and ß2GPI was observed under any experimental conditions. Figure 1D–F reveals the time-dependent formation of oxLDL complexes with CRP and/or ß2GPI in the presence of 1.25 mM of Ca2+ ion at 37°C. The interaction of oxLDL with CRP and oxLDL with ß2GPI occurred in a time-dependent as well as dose-dependent manner. These two interactions progressed gradually and reached a plateau after ~12 h.


Figure 1
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  		Fig. 1. Profiles of complex formation among Cu2+-oxidized LDL (oxLDL), ß2-glycoprotein I (ß2GPI), and C-reactive protein (CRP). For the experiment, oxLDL/ß2GPI and CRP/oxLDL complexes were preformed by incubating Cu2+-oxLDL [1 mg apolipoprotein B (apoB) equivalent/ml] and ß2GPI (1 mg/ml) in the absence of CaCl2, and Cu2+-oxLDL (1 mg apoB equivalent/ml) and CRP (1 mg/ml) in the presence of 2 mM CaCl2, respectively, at 37°C for 16 h. Subsequently, the complexes were purified by size exclusion column chromatography, as shown in Fig. 2B. A–C: CRP, oxLDL, ß2GPI, preformed oxLDL/ß2GPI complexes, and/or preformed CRP/oxLDL complexes were incubated at 37°C for 16 h in the presence of different concentrations of CaCl2. Concentration of each material was 50 µg/ml protein or 50 µg/ml apoB-100 equivalent. Reaction was terminated by immediate freeze, and an aliquot of each specimen was diluted up to 0.5 µg/ml of apoB equivalent (1:100 dilution) to apply to the ELISAs. Generation of oxLDL/ß2GPI complexes (A), CRP/oxLDL complexes (B), or CRP/oxLDL/ß2GPI complexes (C) was detected in ELISAs, as described in Materials and Methods. D–F: CRP, oxLDL, ß2GPI, preformed oxLDL/ß2GPI complexes, and/or preformed CRP/oxLDL complexes were incubated at 37°C for different periods in the presence of 1.25 mM CaCl2, and generated complexes (D–F) were measured by ELISA, as described above. All values in the in vitro experiments are expressed as the mean ± SD. Circles, CRP + oxLDL + ß2GPI; triangles, preformed oxLDL/ß2GPI complexes + CRP; squares, preformed CRP/oxLDL complexes + ß2GPI. Error bars represent mean ± SD.

 

Figure 2
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  		Fig. 2. In vitro formation of CRP/oxLDL/ß2GPI complexes. Cu2+-OxLDL (1 mg/ml of apoB equivalent) was incubated with ß2GPI (1 mg/ml) for 16 h at 37°C to form oxLDL/ß2GPI complexes. The purified oxLDL/ß2GPI (1 mg/ml of apoB equivalent) was further incubated with CRP (purified from human pleural fluid; 0.1 mg/ml) in the presence of 2 mM CaCl2 for 16 h at 37°C, to form nondissociable CRP/oxLDL/ß2GPI complexes. The complexes were purified by size exclusion chromatography. A: Electrophoresis on an agarose gel was performed and proteins were visualized by amido black staining. The negative charge in oxLDL was significantly neutralized by the complex formation with CRP and ß2GPI. B: Elution profile of the size exclusion column chromatography of CRP/oxLDL/ß2GPI complexes is shown. The reaction mixture (100 µl aliquot) containing CRP and oxLDL/ß2GPI complexes was applied on a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) equipped with fast-protein liquid chromatography and eluted with Tris buffer containing 1.25 mM CaCl2. CRP/oxLDL/ß2GPI and oxLDL/ß2GPI complexes in 100-fold diluted fractions were detected by ELISAs, and CRP in nondiluted fractions was detected by high-sensitivity CRP nephelometry.

 
While the interaction between ß2GPI and oxLDL was calcium independent, the interaction between CRP and oxLDL was calcium dependent. At physiological concentrations of calcium, long incubations (of several hours) were required for the in vitro formation of nondissociable CRP/oxLDL/ß2GPI complexes or oxLDL/ß2GPI complexes. The oxLDL-negative charges acquired during CuSO4 incubation were neutralized by the interaction with these proteins (Fig. 2A ).

We also confirmed that significant in vitro production of CRP/oxLDL/ß2GPI complexes occurs at reduced endotoxin concentrations (28 pg/ml). However, the potential influence of such small amounts of endotoxin contaminating the reaction mixture on complex formation should be investigated in further studies.

Figure 2B shows the elution profile of size exclusion chromatography of the reaction mixture containing CRP and oxLDL/ß2GPI complexes. CRP/oxLDL/ß2GPI and oxLDL/ß2GPI complexes were mainly eluted in fractions 17–30 (mainly 17–22) and 17–30 (mainly 20–30), respectively. CRP was detected in the first peak (corresponding to the size of CRP/oxLDL/ß2GPI particles) and in a later fraction (38–42), where the commercially available pentameric CRP (120 kDa; derived from pleural fluid or serum) was eluted. The contamination of the modified CRP isoform having a molecular mass of 75 kDa (38) in the CRP protein(s) eluted in fractions larger than 50 kDa cannot be excluded. Thus, these results indicate that CRP/oxLDL/ß2GPI complexes can cross-react in the hsCRP assay. The total recovery of CRP from all eluates (including noncomplexed CRP) detected by hsCRP nephelometry varied in individual experiments, with ranges between 38% and 70%.

Diagnostic accuracy of CRP/oxLDL/ß2GPI complex assay
The principle of the ELISA for CRP/oxLDL/ß2GPI complexes is schematically represented in Fig. 3A . The assay was specific for CRP/oxLDL/ß2GPI complexes without any cross-reactivity to CRP/oxLDL complexes or oxLDL/ß2GPI complexes (Fig. 3B). In addition, the CRP/oxLDL/ß2GPI complex value (30 ng/ml of apoB equivalent) was not affected by addition of excess amounts of oxLDL/ß2GPI complexes (300 ng/ml of apoB equivalent, expected as pathophysiological concentration) in the CRP/oxLDL/ß2GPI ELISA. The ELISA for oxLDL/ß2GPI complexes was previously described elsewhere (10).


Figure 3
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  		Fig. 3. Sandwich ELISA for oxLDL/ß2GPI complexes. Assay principle of the ELISA (A) and specificity (cross-reactivity) (B) are shown. Circles, CRP/oxLDL/ß2GPI; squares, oxLDL/ß2GPI; triangles, CRP/oxLDL.

 
In the present study, the clinical significance of circulating CRP/oxLDL/ß2GPI, oxLDL/ß2GPI, and CRP/oxLDL complexes was assessed by ELISA. Cutoff values for these ELISAs were analyzed by two methods: a receiver operating characteristic (ROC) curve using DM patients, and by calculating one to five standard deviations (SDs) above mean values using 48 healthy subjects. For the ROC analysis, atherosclerosis was diagnosed according to IMT measurements. A mean + 3 SD of the healthy subjects was used as cutoff value. One U/ml was defined as the mean + 3 SD of controls. The assay was reproducible, with intra- and inter-assay coefficients of variation not exceeding 7.0%. The same procedure was used to calculate the CRP cutoff value (0.16 mg/dl).

Diagnostic accuracy of the CRP/oxLDL/ß2GPI complex ELISA for atherosclerosis, as compared with CRP nephelometry and the other two complex ELISAs, was assessed by determining sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), odds ratio (OR), and 95% confidence interval (CI) (Table 1 ). ROC curves for these three markers were similar (data not shown). In the present study, a subgroup of 69 DM patients (49 patients without atherosclerosis and 20 patients with atherosclerosis) was analyzed. Both CRP/oxLDL/ß2GPI and CRP/oxLDL ELISAs showed lower sensitivity (25%) but 100% specificity and PPV, and similar NPV (76.6%) compared with the CRP assay. The measurement of circulating CRP/oxLDL/ß2GPI complexes may reliably help to predict the development of atherosclerosis. Thus, a major benefit of these complex ELISAs is that they allow the exclusion of cross-reactive noncomplexed CRP isoforms frequently found in sera of patients with various unrelated acute/chronic infections.


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  		TABLE 1. Diagnostic accuracy of CRP and oxLDL complexes

 
Serum levels of oxLDL/ß2GPI and CRP/oxLDL/ß2GPI complexes
Figure 4 shows the serum levels of CRP, oxLDL/ß2GPI, CRP/oxLDL, and CRP/oxLDL/ß2GPI complexes in patients with various diseases and in healthy subjects. Elevated levels of CRP were observed in patients with DM, RA, and pyrogenic diseases [25.6% (32/125), 75.0% (36/48), and 100% (35/35), respectively] (Fig. 4A). Statistical significance was observed between DM and healthy subjects, RA and healthy subjects, and DM and RA patients. OxLDL/ß2GPI complexes were found in 44.0% of DM patients (55/125) but not in healthy subjects, RA patients, or patients with pyrogenic diseases (Fig. 4B). Seven DM patients had high levels of CRP/oxLDL (5.6%) and nine had high levels of CRP oxLDL/ß2GPI complexes (7.2%). Five DM patients had both (Fig. 4C, D). Although none of the patients in the other groups showed high levels of CRP/oxLDL/ß2GPI complexes, two RA patients had slightly elevated levels of CRP/oxLDL complexes (just above the cutoff value). CRP/oxLDL/ß2GPI and CRP/oxLDL complex levels between DM and healthy subjects, DM and RA, and DM and patients with pyrogenic diseases were statistically significant.


Figure 4
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  		Fig. 4. Serum levels of CRP and three kinds of oxLDL complexes in patients with diabetes mellitus (DM), rheumatoid arthritis (RA), or pyrogenic disease. Serum CRP was measured by the routine or high-sensitivity methods (A). Serum oxLDL/ß2GPI complexes (B), CRP/oxLDL complexes (C), and CRP/oxLDL/ß2GPI complexes (D) were detected by ELISA. Complex levels are expressed in arbitrary units. A sample was considered positive when its complex level was higher than the cutoff value, 1 U/ml. The number of patients and mean values for each group are shown. The Student's t-test was performed. P < 0.05 was considered statistically significant. NS, not significant.

 
Sixty-nine DM patients, a subpopulation of 125 DM patients described in Materials and Methods, were divided into two groups according to the presence (AT+) or absence (AT–) of atherosclerosis (AT+ with IMT >0.75 mm, n = 20 and AT– with IMT <0.75 mm, n = 49). Serum levels of CRP, CRP/oxLDL and CRP/oxLDL/ß2GPI complexes but not oxLDL/ß2GPI complexes were significantly higher in AT+ than those in AT– (Fig. 4).

There was no correlation between serum levels of oxLDL/ß2GPI and CRP/oxLDL/ß2GPI complexes (Fig. 5A ). In contrast, strong correlation between CRP and CRP/oxLDL/ß2GPI complexes (Fig. 5B), CRP and CRP/oxLDL (Fig. 5C), and CRP/oxLDL and CRP/oxLDL/ß2GPI (Fig. 5D) were observed in DM patients. Thus, CRP/oxLDL and CRP/oxLDL/ß2GPI found in DM patients may be a part of the hsCRP currently used as a predictive marker for CVD. Further, these correlations seem to indicate that CRP/oxLDL complexes in DM patients were mostly complexed with ß2GPI.


Figure 5
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  		Fig. 5. Correlation among serum levels of CRP/oxLDL/ß2GPI complexes, oxLDL/ß2GPI, CRP/oxLDL complexes, and CRP. Serum levels of these complexes and CRP in individual DM patients are indicated. (A) Correlation between OXLDL/ß2GPI and CRP/OXLDL/ß2GPI complexes; (B) correlation between CRP and CRP/OXLDL complexes; (C) correlation between CRP and CRP/OXLDL/ß2GPI complexes; (D) correlation between CRP/OXLDL and CRP/OXLDL/ß@GPI complexes. The correlation between two variables was evaluated by the Pearsons' correlation test.

 
Detection of CRP/oxLDL/ß2GPI complexes in serum samples by immunoblot analysis
To confirm the presence of oxLDL complexes containing CRP and ß2GPI, nondenaturing PAGE (native PAGE without SDS)/immunoblot analysis was performed in serum samples from healthy subjects, CRP/oxLDL/ß2GPI complex-positive DM patients with atherosclerosis, CRP-positive RA patients, and CRP-positive patients with pyrogenic diseases (seven samples from each group). Typical immunoblot patterns from two samples of each group are shown in Fig. 6 . Two noncomplexed CRP isoforms were detected in DM and RA patients and those with pyrogenic diseases. In contrast, noncomplexed ß2GPI was detected in all serum samples. The complex forms of LDL with CRP and of ß2GPI were only detected in the DM sera (at the upper position of the LDL band). As described in Materials and Methods, the gels were run at pH 8.3, much closer to the reported pI of CRP (5.3 and 7.4) (39) and ß2GPI (5.0 to 7.0) (40) than those of molecular markers (4.0 to 4.7) used in this experiment. Actually, mobility of CRP and ß2GPI in agarose gel electrophoresis (run at pH 8.6) was relatively smaller than that of BSA (pI 4.7)(Fig. 2A). Therefore, molecular sizes of pentameric CRP (120 kDa) and ß2GPI (50 kDa) determined by nondenaturing PAGE in this study were not consistent with previous reports.


Figure 6
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  		Fig. 6. Immunoblot analysis of oxLDL complexes in serum samples. All proteins were stained in the left panel. Transferred proteins onto a PVDF membrane were reacted with anti-CRP Ab, anti-b2GPI Ab, or anti-apoB100 Ab. Serum samples from healthy subjects (lanes 1 and 2), DM patients (lanes 3 and 4), RA patients (lanes 5 and 6), or patients with pyrogenic disease (lanes 7 and 8) were run on the nondenaturing PAGE (native PAGE without SDS). The proteins were transferred onto the polyvinylidene difluoride membrane, and immunoblot analysis was performed with rabbit anti-CRP antiphospholipid antibodies, mouse anti-ß2GPI monoclonal antibody (MAb), Cof-23, or mouse anti-apoB100 MAb, N2E10. The arrows indicate positions of marker proteins (on the left side) and of LDL, noncomplexed ß2GPI or noncomplexed CRP isoforms (on the right side).

 
Co-localization of ß2GPI, CRP, and oxLDL in macrophages and smooth muscle cells from atherosclerotic lesions
We investigated the localization of ß2GPI, CRP, and oxLDL in atherosclerotic carotid arteries and RA synovial tissues by immunohistochemistry. As shown in Fig. 7 , clusters of foamy macrophages and layered smooth muscle cells with lipid droplets were commonly observed in cartoid endarterectomy specimens (Fig. 7A, B). These foamy macrophages and smooth muscle cells strongly expressed scavenger receptors for oxLDL (SR-PSOX) (Fig. 7C). Furthermore, CRP (Fig. 7D), apoB and oxLDL (Fig. 7E, F, respectively), noncomplexed/complexed ß2GPI (Fig. 7G), and the complexed form of ß2GPI (Fig. 7H) were always detected in these macrophages and smooth muscle cells. The presence of ß2GPI in carotid atherosclerotic plaques is consistent with the previous report by George et al. (41).


Figure 7
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  		Fig. 7. Immunostaining of carotid atherosclerotic lesions (original magnification: x60). Round foamy macrophages were observed in the lower half of the slides (A) (CD68), whereas spindle-shaped and foamy smooth muscle cells existed in the upper half (B) (SMC{alpha}). Staining of serial sections indicated that almost all macrophages and smooth muscle cells expressed SR-PSOX (C), CRP (D), apoB (E), oxLDL (F), ß2GPI (G), and the complexed form of ß2GPI (H).

 
Macrophage infiltration was also commonly observed in RA synovium (Fig. 8A ) and colocalized with oxLDL (Fig. 8C), and some macrophages obviously showed CRP immunoreactivity (Fig. 8D). Interestingly, no expression of SR-PSOX (Fig. 8B) and no presence of ß2GPI (Fig. 8E) was observed. In contrast, oxLDL, CRP, and ß2GPI were not detected in osteoarthritis synovial tissues (data not shown).


Figure 8
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  		Fig. 8. Immunostaining of RA synovium (original magnification: x100). Severe infiltration of macrophages was observed in RA synovial tissue (A) (CD68). oxLDL (C) and CRP (D) were detected in these cells. Positive staining of SR-PSOX (B) and the complexed form of ß2GPI (E) was not obtained in the synovial macrophages.

 
Association with biological markers related to atherosclerosis
To further evaluate the implication of CRP and these oxLDL complexes in atherosclerosis, the association with several biological serum markers was analyzed. As shown in Table 2 , serum CRP levels were positively associated with those of sICAM-1 and sVCAM-1. In contrast, oxLDL/ß2GPI complexes were associated with T-chol and HbA1c. However, there was no association between CRP/oxLDL/ß2GPI complexes and any markers determined in this study. The results support the previous observations that CRP activates endothelial cells (29) and that oxidative stress (i.e., oxidation of LDL and complex formation) in the intima is related to hypercholesterolemia and hyperglycemia.


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  		TABLE 2. Association between CRP, three types of oxLDL complexes, and biological markers in DM patients

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have recently demonstrated that oxLDL interacts with ß2GPI and the presence of oxLDL/ß2GPI complexes circulating in patients with atherosclerotic and inflammatory diseases, such as systemic lupus erythematosus, APS, DM, and chronic nephritis (4, 6, 10). High levels of oxLDL/ß2GPI complexes were also present in atherosclerosis-prone mice with apoe–/– and ldlr–/– genotypes, especially those fed a high-cholesterol diet (unpublished observations). These observations strongly suggested that increased serum levels of oxLDL/ß2GPI complexes could represent a novel and clinically useful serologic marker for the assessment of atherosclerosis. In this study, we found CRP/oxLDL/ß2GPI (and CRP/oxLDL) as well as oxLDL/ß2GPI complexes in sera of patients with DM. CRP/oxLDL/ß2GPI (and CRP/oxLDL) complexes were particularly present in patients with IMT-diagnosed atherosclerosis, and their levels strongly correlated with hsCRP but not with oxLDL/ß2GPI complexes. Interestingly, LDL complexes containing ß2GPI were not present in sera of patients with RA or pyrogenic diseases. Routine CRP/hsCRP nephelometry for diagnosing CVD may provide false-positive results. In contrast, in the present study, the CRP/oxLDL/ß2GPI complex ELISA showed 100% specificity and positive predictive value due to the lack of reactivity of samples from pyrogenic diseases and RA.

We postulate that CRP/oxLDL/ß2GPI complexes are mainly, or possibly, only formed in atherosclerotic lesions based on the following observations: Immunohistochemistry of carotid artery plaques showed colocalization of oxLDL, ß2GPI, and CRP with SR-PSOX-positive foamy macrophages and activated/transformed smooth muscle cells. Our previous (10) and present in vitro studies actually demonstrated a stoichiometric interaction between Cu2+-oxLDL and ß2GPI and that stable/non-dissociable complexes of oxLDL/ß2GPI and CRP/oxLDL/ß2GPI were gradually generated up to 24 h of incubation at 37°C (Fig. 1D–F). OxLDL injected intravenously into experimental animals was quickly removed from the circulation by the liver (half life ~10 min), due to its negative charge (17). Even though high concentrations of ß2GPI (about 200 µg/ml) are present in the circulation, it seems impossible that oxLDL could form complexes with circulating ß2GPI in such a short period of time. In contrast, a 6-month follow-up study of DM patients indicated that serum levels of these complexes were very steady (data not shown). Therefore, CRP/oxLDL/ß2GPI and oxLDL/ß2GPI complexes found in the circulation were probably generated in atherosclerotic lesions, not in the blood stream.

It is now widely accepted that CRP contributes to arterial inflammation and pro-atheroscelerotic phenotypes by upregulating adhesion molecules of endothelial cells, such as ICAM-1 and VCAM-1 (42). Dyslipoproteinemia and hyperglycemia contribute to oxidation of LDL, which leads to the progression of atherosclerosis via different mechanisms by activating or damaging endothelial cells with the participation of CRP (43). Two positive associations were observed in the present study: one between serum CRP and sICAM-1/sVCAM-1 levels, and another between oxLDL/ß2GPI complex and T-chol/HbA1c levels, both supporting the mechanisms mentioned above. As shown in Table 2, there were no significant associations of serum CRP/oxLDL/ß2GPI complexes with these pro-atherosclerotic parameters, because CRP/oxLDL/ß2GPI complexes may represent a unique combination and a novel marker for the assessment of atherosclerosis severity. These complexes are formed by two distinct mechanisms: CRP generated from vascular inflammation, and oxLDL (oxLDL/ß2GPI) generated from oxidative stress within the intima.

The interaction of CRP with oxLDL is considered a key event linked to atherosclerosis. It has been recently reported that monomeric or structurally modified (not pentameric) CRP isoform(s) can bind to apoB and lipid ligands either on native LDL or oxLDL in a calcium-dependent manner (24). Interestingly, our native PAGE/immunoblot analysis also revealed the presence of two noncomplexed CRP isoforms in all CRP/oxLDL/ß2GPI complexes-positive DM sera, and one of them may be a spontaneously dissociated mononeric form and/or a structurally modified form (Fig. 6). Another possibility is that the doublet bands come from the pentraxin form of CRP and have different pIs because of their different conformation, as previously reported (39). In contrast, as we previously described elsewhere, the {omega}-carboxyl function at the acyl chain of cholesteryl esters in oxLDL was responsible for the initial ß2GPI binding (4, 8–10). The carboxyl function is a late oxidative product that appears after severe lipid peroxidation of LDL, but not in minimally modified LDL. Taken together, these results indicate that circulating CRP/oxLDL/ß2GPI complexes are most probably generated from highly oxidized LDL in atherosclerotic lesions but not from native or minimally modified LDL.

As shown in the present study, significant amounts of noncomplexed isoforms of CRP were detected in sera of DM patients with atherosclerosis, as well as CRP/oxLDL/ß2GPI complexes (immunoblot analysis in Fig. 6). It is generally thought that circulating CRP comes from the liver and that the hepatic production of CRP is mainly upregulated by inflammatory cytokines, such as interleukin-6. However, it has been recently reported that CRP can also be produced locally in atherosclerotic lesions by endothelial cells, smooth muscle cells, and monocytic cells (44–46). It is still under discussion, but CRP as an autocrine and a paracrine factor probably promotes atherosclerosis by interfering with endothelial cell regulation, altering vascular smooth muscle cell and/or monocyte/macrophage functions (29). As described herein, ß2GPI complexes with oxLDL or CRP/oxLDL are most probably generated only in atherosclerotic lesions; however, it is still unclear whether oxLDL is complexed with liver or locally generated CRP.

Finally, atherosclerotic complications have been described in RA patients, but ß2GPI complexes with oxLDL or CRP/oxLDL complexes were not detected in the sera from these patients. We demonstrated that oxLDL, ß2GPI, and CRP were colocalized with SR-PSOX-positive foamy macrophages and activated or transformed smooth muscle cells in carotid artery plaques, but not with ß2GPI in RA synovial tissues. In addition, synovial macrophages did not express scavenger receptor SR-PSOX, nor did they develop into foam cells. This can be interpreted as indicating that synovial macrophages are not sufficiently activated. LDL in RA synovial fluid was slightly more electronegative than LDL from matched plasma samples, possibly representing minimally modified LDL (47, 48). Because changes of minimally modified LDL are much less pronounced than those produced by Cu2+ treatment, the finding of minimal cholesterol or cholesteryl ester loading in synovial macrophages was expected. The degree of LDL oxidation and macrophage activation in carotid atherosclerotic plaques, unlike that in synovial tissue, is strong enough to cause the binding of ß2GPI. Thus, the development of atherosclerosis in DM patients may occur as a result of a mechanism somewhat different from that in RA patients.

In conclusion, oxLDL complexes containing both ß2GPI and CRP are formed under inflammatory and oxidative stress conditions in atherosclerotic lesions and are released into the circulation as electrostatically neutral and stable complexes. Determination of circulating CRP/oxLDL/ß2GPI complexes may be useful in assessing the development of atherosclerosis and/or in diagnosing atherosclerosis. However, the clinical significance of these novel markers (CRP/oxLDL/ß2GPI and oxLDL/ß2GPI) as well as CRP/oxLDL should be further elucidated in larger clinical studies.


   ACKNOWLEDGMENTS
 
This study was supported in part by a research grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and by a research grant from the Ministry of Health, Labor and Welfare of Japan. The authors thank Dr. Dennis R. Voelker (National Jewish Medical and Research Center, Denver, CO) for helpful discussions.

Manuscript received September 18, 2006 and in revised form January 16, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
  1. Steinberg, D. 1997. Low density lipoprotein oxidation and its pathobiological significance. J. Biol. Chem. 272: 20963–20966.[Free Full Text]

  2. Heinecke, J. W. 1997. Mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis. Curr. Opin. Lipidol. 8: 268–274.[Medline]

  3. Hughes, G. R. V., E. N. Harris, and A. E. Gharavi. 1986. Anti-cardiolipin syndrome. J. Rheumatol. 13: 486–489.[Medline]

  4. Matsuura, E., K. Kobayashi, M. Tabuchi, and L. R. Lopez. 2006. Oxidative modification of low-density lipoprotein and immune regulation of atherosclerosis. Prog. Lipid Res. 45: 466–486.[CrossRef][Medline]

  5. Matsuura, E., Y. Igarashi, T. Yasuda, D. A. Triplett, and T. Koike. 1994. Anticardiolipin antibodies recognize ß2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J. Exp. Med. 179: 457–462.[Abstract/Free Full Text]

  6. Kasahara, H., E. Matsuura, K. Kaihara, D. Yamamoto, K. Kobayashi, J. Inagaki, K. Ichikawa, A. Tsutsumi, S. Yasuda, T. Atsumi, et al. 2005. Antigenic structures recognized by anti-ß2-glycoprotein I auto-antibodies. Int. Immunol. 17: 1533–1542.[Abstract/Free Full Text]

  7. Hasunuma, Y., E. Matsuura, Z. Makita, T. Katahira, S. Nishi, and T. Koike. 1997. Involvement of ß2-glycoprotein I and anticardiolipin antibodies in oxidatively modified low-density lipoprotein uptake by macrophages. Clin. Exp. Immunol. 107: 569–573.[CrossRef][Medline]

  8. Kobayashi, K., E. Matsuura, Q. Liu, J. Furukawa, K. Kaihara, J. Inagaki, T. Atsumi, N. Sakairi, T. Yasuda, D. R. Voelker, et al. 2001. A specific ligand for ß2-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages. J. Lipid Res. 42: 697–709.[Abstract/Free Full Text]

  9. Liu, Q., K. Kobayashi, J. Furukawa, J. Inagaki, N. Sakairi, A. Iwado, T. Yasuda, T. Koike, D. R. Voelker, and E. Matsuura. 2002. {omega}-Carboxyl variants of 7-ketocholesteryl esters are ligands for ß2-glycoprotein I and mediate antibody-dependent uptake of oxidized LDL by macrophages. J. Lipid Res. 43: 1486–1495.[Abstract/Free Full Text]

  10. Kobayashi, K., M. Kishi, T. Atsumi, M. L. Bertolaccini, H. Makino, N. Sakairi, I. Yamamoto, T. Yasuda, M. A. Khamashta, G. R. V. Hughes, et al. 2003. Circulating oxidized LDL forms complexes with ß2-glycoprotein I: implication as an atherogenic autoantigen. J. Lipid Res. 44: 716–726.[Abstract/Free Full Text]

  11. Shoenfeld, Y., R. Wu, L. D. Dearing, and E. Matsuura. 2004. Are anti-oxidized low-density lipoprotein antibodies pathogenic or protective? Circulation. 110: 2552–2558.[Free Full Text]

  12. Shoenfeld, Y., R. Gerli, A. Doria, E. Matsuura, M. M. Cerinic, N. Ronda, L. J. Jara, M. Abu-Shakra, P. L. Meroni, and Y. Sherer. 2005. Accelerated atherosclerosis in autoimmune rheumatic diseases. Circulation. 112: 3337–3347.[Free Full Text]

  13. Lopez, L. R., B. L. Hurley, D. F. Simpson, and E. Matsuura. 2005. Oxidized low-density lipoprotein/ß2-glycoprotein I complexes and autoantibodies in patients with type 2 diabetes mellitus. Ann. N. Y. Acad. Sci. 1051: 97–103.[Abstract/Free Full Text]

  14. Kasahara, J., K. Kobayashi, Y. Maeshima, Y. Yamasaki, T. Yasuda, E. Matsuura, and H. Makino. 2004. Clinical significance of serum oxidized low-density lipoprotein/ß2-glycoprotein I complexes in patients with chronic renal diseases. Nephron Clin. Pract. 98: c15–c24.

  15. Lopez, D., I. Garcia-Valladares, C. A. Palafox-Sanchez, I. G. De La Torre, K. Kobayashi, E. Matsuura, and L. R. Lopez. 2004. Oxidized low-density lipoprotein/ß2-glycoprotein I complexes and autoantibodies to oxLig-1/ß2-glycoprotein I in patients with systemic lupus erythematosus and antiphospholipid syndrome. Am. J. Clin. Pathol. 121: 426–436.[CrossRef][Medline]

  16. Lopez, L. R., M. Salazar-Paramo, C. Palafox-Sanchez, B. L. Hurley, E. Matsuura, and I. Garcia-De La Torre. 2006. Oxidized low-density lipoprotein and ß2-glycoprotein I in patients with systemic lupus erythematosus and increased carotid intima-media thickness: implications in autoimmune-mediated atherosclerosis. Lupus. 15: 80–86.[Abstract/Free Full Text]

  17. Van Berkel, T. J., Y. B. De Rijke, and J. K. Kruijt. 1991. Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells. J. Biol. Chem. 266: 2282–2289.[Abstract/Free Full Text]

  18. Volanakis, J. E. 2001. Human C-reactive protein: expression, structure, and function. Mol. Immunol. 38: 189–197.[CrossRef][Medline]

  19. Paffen, E., and M. P. Demaat. 2006. C-reactive protein in atherosclerosis: a causal factor? Cardiovasc. Res. 71: 30–39.[Abstract/Free Full Text]

  20. Pepys, M. B., and G. M. Hirschfield. 2003. C-reactive protein: a critical update. J. Clin. Invest. 111: 1805–1812.[CrossRef][Medline]

  21. Chang, M-K., C. J. Binder, M. Torzewski, and J. L. Witztum. 2002. C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand: phosphorylcholine of oxidized phospholipids. Proc. Natl. Acad. Sci. USA. 99: 13043–13048.[Abstract/Free Full Text]

  22. Binder, C. J., S. Horkko, A. Dewan, M. K. Chang, E. P. Kieu, C. S. Goodyear, P. X. Shaw, W. Palinski, J. L. Witztum, and S. G. Jilverman. 2003. Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat. Med. 9: 736–743.[CrossRef][Medline]

  23. Mold, C. 1999. Role of complement in host defense against bacterial infection. Microbes Infect. 1: 633–638.[CrossRef][Medline]

  24. Ji, S-R., Y. Wu, L. A. Potempa, Q. Qiu, and J. Zhao. 2006. Interactions of C-reactive protein with low-density lipoproteins: implications for an active role of modified C-reactive protein in atherosclerosis. Int. J. Biochem. Cell Biol. 38: 648–661.[CrossRef][Medline]

  25. Libby, P., and P. M. Ridker. 2004. Inflammation and atherosclerosis: role of C-reactive protein in risk assessment. Am. J. Med. 116 (Suppl. 6A): 9–16.[CrossRef]

  26. Verma, S., P. E. Szmitko, and P. M. Ridker. 2005. C-reactive protein comes of age. Nat. Clin. Pract. Cardiovasc. Med. 2: 29–36.[CrossRef][Medline]

  27. Pasceri, V., J. T. Willerson, and E. T. H. Yeh. 2000. Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation. 102: 2165–2168.[Abstract/Free Full Text]

  28. Pasceri, V., J. S. Cheng, J. T. Willerson, and E. T. H. Yeh. 2001. Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs. Circulation. 103: 2531–2534.[Abstract/Free Full Text]

  29. Verma, S., S. Devaraj, and I. Jialal. 2006. Is C-reactive protein an innocent bystander or proatherogenic culprit? C-reactive protein promotes atherothrombosis. Circulation. 113: 2135–2150.[Medline]

  30. WHO Study Group. 1985. Diabetes mellitus. World Health Organ. Tech. Rep. Ser. 727: 1–113.

  31. Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries, N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, et al. 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31: 315–324.[Medline]

  32. Hashimoto, Y., M. Kawamura, K. Ichikawa, T. Suzuki, T. Sumida, S. Yoshida, E. Matsuura, S. Ikehara, and T. Koike. 1992. Anticardiolipin antibodies in NZW x BXSB F1 mice. A model of antiphospholipid syndrome. J. Immunol. 149: 1063–1068.[Abstract]

  33. Igarashi, M., E. Matsuura, Y. Igarashi, H. Nagae, K. Ichikawa, D. A. Triplett, and T. Koike. 1996. Human ß2-glycoprotein I as an anticardiolipin cofactor determined using mutants expressed by a baculovirus system. Blood. 87: 3262–3270.[Abstract/Free Full Text]

  34. Minami, M., N. Kume, T. Shimaoka, H. Kataoka, K. Hayashida, Y. Akiyama, I. Nagata, K. Ando, M. Nobuyoshi, M. Hanyuu, et al. 2001. Expression of SR-PSOX, a novel cell-surface scavenger receptor for phosphatidylserine and oxidized LDL in human atherosclerotic leisions. Arterioscler. Thromb. Vasc. Biol. 21: 1796–1800.[Abstract/Free Full Text]

  35. Krauss, R. M., and D. J. Burke. 1982. Identification of multiple subclasses of plasma low density lipoproteins in normal humans. J. Lipid Res. 23: 97–104.[Abstract]

  36. Ozaki, K., K. Inoue, H. Sato, A. Iida, Y. Ohnishi, A. Sekine, H. Sato, K. Odashiro, M. Nobuyoshi, M. Hori, et al. 2004. Functional variation in LGALS2 confers risk of myocardial infarction and regulates lymphotoxicin-{alpha} secretion in vitro. Nature. 429: 72–75.[CrossRef][Medline]

  37. Shi, S-R., M. E. Key, and K. L. Kalra. 1991. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating tissue sections. J. Histochem. Cytochem. 39: 741–748.[Abstract]

  38. Devaraj, S., S. Venugopal, and I. Jialal. 2006. Native pentameric C-reactive protein displays more potent pro-atherogenic activities in human aortic endothelial cells than modified C-reactive protein. Atherosclerosis. 184: 48–52.[CrossRef][Medline]

  39. Tsujimoto, M., K. Inoue, and S. Nojima. 1983. Purification and characterization of human serum C-reactive protein. J. Biochem. (Tokyo). 94: 1367–1373.[Abstract/Free Full Text]

  40. Buttari, B., E. Profumo, V. Mattei, A. Siracusano, E. Ortona, P. Margutti, B. Salvati, M. Sorice, and R. Rigano. 2005. Oxidized ß2-glucoprotein I induces human dendritic cell maturation and promotes a T helper type 1 response. Blood. 106: 3880–3887.[Abstract/Free Full Text]

  41. George, J., D. Harats, B. Gilburd, A. Afek, Y. Levy, J. Schneiderman, I. Barshack, J. Kopolovic, and Y. Shoenfeld. 1999. Immunolocalization of ß2-glycoprotein I (apolipoprotein H) to human atherosclerotic plaques: potential implications for lesion progression. Circulation. 99: 2227–2230.[Abstract/Free Full Text]

  42. Pasceri, V., H. D. Wu, J. T. Willerson, and E. T. H. Yeh. 2000. Modulation of vascular inflammation in vitro and in vivo by peroxisome proliferator-activated receptor-gamma activators. Circulation. 101: 235–238.[Abstract/Free Full Text]

  43. Steinberg, D., and A. Lewis. 1997. Conner memorial lecture. Oxidative modification of LDL and atherosclerosis. Circulation. 95: 1062–1071.[Free Full Text]

  44. Venugopal, S. K., S. Devaraj, and I. Jialal. 2005. Macrophage conditioned medium induces the expression of C-reactive protein in human aortic endothelial cells: potential for paracrine/autocrine effects. Am. J. Pathol. 166: 1265–1271.[Abstract/Free Full Text]

  45. Yasojima, K., C. Schwab, E. G. McGeer, and P. L. McGeer. 2001. Generation of C-reactive protein and complement components in atherosclerotic plaques. Am. J. Pathol. 158: 1039–1051.[Abstract/Free Full Text]

  46. Calabro, P., J. T. Willerson, and E. T. H. Yeh. 2003. Inflammatory cytokines stimulated C-reactive protein production by human coronary artery smooth muscle cells. Circulation. 108: 1930–1932.[Abstract/Free Full Text]

  47. James, M. J., D. van Reyk, K. A. Rye, R. T. Dean, L. G. Cleland, P. J. Barter, and W. Jessup. 1998. Low density lipoprotein of synovial fluid in inflammatory joint disease is mildly oxidized. Lipids. 33: 1115–1121.[Medline]

  48. Dai, L., Z. Zhang, P. G. Winyard, K. Gaffney, H. Jones, D. R. Blake, and C. J. Morris. 1996. A modified form of low-density lipoprotein with increased electronegative charge is present in rheumatoid arthritis synovial fluid. Fee Radic. Res. 22: 705–710.


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