Published online before print August 15, 2002, doi: 10.1161/01.ATV.0000033829.14012.18
© 2002 American Heart Association, Inc.
Atherosclerosis |
Oxidized LDL in Carotid Plaques and Plasma Associates With Plaque Instability
From the Department of Neurosurgery (K.N., M.U., K.T.K., K.S., S.N.) and Pathology (H.H.), School of Medicine, The University of Tokushima, Tokushima, Japan, and Department of Molecular Pathology (H.I.), Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Correspondence to Masaaki Uno, MD, Department of Neurosurgery, School of Medicine, The University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan. E-mail muno{at}clin.med.tokushima-u.ac.jp
Abstract
Objective— Oxidation of LDL plays a significant pathogenic role in atherosclerosis. In this study, we attempted to clarify the correlation between the morphology of human atherosclerotic plaques and the oxidized LDL (OxLDL) levels in plasma and carotid plaques.
Methods and Results— OxLDL levels (ng/µg apolipoprotein B) in plasma and carotid plaques from 44 patients undergoing carotid endarterectomy and OxLDL levels in 17 control plasma and 9 normal intima samples were determined by a sandwich ELISA by using specific antibodies against OxLDL (DLH3) and apolipoprotein B. The plaques were immunohistochemically classified as macrophage (M
)-rich and M-poor. In paired samples from individual patients, plaque OxLDL was nearly 70 times higher than plasma OxLDL (mean±SEM, 11.9±1.7 vs 0.18±0.01 ng/µg apoB, P<0.0001). The OxLDL level was significantly higher in M-rich- than M-poor plaques (19.6±2.8 vs 5.50±0.77ng/µg apoB, P<0.0001) and corresponded with DLH3 antigen positivity of the plaques. In patients with M-rich plaques, plasma OxLDL was significantly higher than in the controls (0.20±0.02 vs 0.13±0.01ng/µg apoB, P=0.02).
Conclusions— Our results suggest that LDL undergoes further oxidation in plaques, and that high plasma and plaque levels of OxLDL are correlated with the vulnerability to rupture of atherosclerotic lesions.
Key Words: oxidized LDL • plaque • plasma • carotid endarterectomy • histopathology
The oxidation of LDL plays a significant pathogenic role in atherosclerosis.1–4 Oxidized LDL (OxLDL) exhibits a number of specific biological properties in vitro and in vivo, including foam cell formation from macrophages. LDL oxidation is a complex process, and a number of oxidation products derived from lipids are generated through peroxidation and fragmentation. Apolipoprotein B (apoB) is exposed to oxidative degradation and modification by oxidized lipids; these processes result in substantially modified lipid and protein components. However, because OxLDL consists of a group of heterogeneously modified particles, it has been difficult to examine OxLDL epitopes in vivo. Itabe et al5 reported a sensitive method to quantify OxLDL using a specific antibody to OxLDL. Oxidized phosphatidylcholine (OxPC) molecules that form adducts with apoB in OxLDL are recognized by the anti-OxLDL monoclonal antibody (mAb), DLH3.6 Using this method, Toshima et al7 and Ehara et al8,9 demonstrated significant increases in the plasma OxLDL levels of patients with coronary heart disease. In addition, previous clinical and experimental studies detected oxidative epitopes in the arterial wall6,8–12; however, to our knowledge, no quantitative analysis of OxLDL in human atherosclerotic lesions has been reported to date.
To clarify how much OxLDL is present and how it is modified in plaques compared with plasma, we assayed the amount of OxLDL in plasma and carotid plaques from patients scheduled for carotid endarterectomy (CEA). We then investigated the relationship between plaque and plasma OxLDL levels and plaque morphology, neurological symptoms, risk factors, and plasma lipid parameters. In our previous study,13 we demonstrated that plaques in which more than 5% of the total area is occupied by macrophages manifested significant elevation of thiobarbituric acid-reactive substances, that they were prone to rupture or fibrous cap thinning, and that they had a large lipid core (
10% of total area). Based on these observations, we considered macrophage infiltration a landmark for plaque instability and classified carotid plaques as macrophage (M)-rich and (M)-poor. We hypothesized that OxLDL level and the degree of macrophage infiltration play a role in whether atherosclerotic lesions are stable or unstable.
Methods
Subjects
Informed consent was obtained from all study participants. Carotid plaque and plasma samples from 44 patients who underwent CEA between June 8, 1999, and December 12, 2000, were examined. All CEAs were performed according to established criteria.14–16 Table 1 summarizes demographic and clinical data on the study participants. Sixteen patients with hyperlipidemia had received lipid-lowering therapy. The side ipsilateral to the operated side was involved in all patients (n=19) who manifested ischemic symptoms (transient ischemic attack, amaurosis fugax, or hemispheric stroke) within 3 months of onset. All operations were performed at the chronic stage, more than one month after onset. The asymptomatic group (n=25) included patients with progression of ipsilateral carotid stenosis confirmed by magnetic resonance angiography or ultrasonography, patients with carotid bruit, and patients who were diagnosed incidentally on medical examination for other complaints (ischemic heart disease or arteriosclerosis obliterans). Control plasma was obtained from 17 age-matched normal volunteers, they were 9 men and 8 women aged 49 to 74 years (mean±SD, 63.3±8.2) without a history of ischemic vascular disease or risk factors. In addition, 9 carotid arteries were removed at autopsy manifested no macroscopic evidence of atherosclerosis. The intima was dissected carefully for examination. CEA specimens were removed en bloc. Immediately after removal, the plaques were examined macroscopically. The thickest part of each plaque was cut transversely to define histomorphological characteristics. The remainder of each plaque was subjected to quantitative analysis of OxLDL. To determine plasma OxLDL, venous blood was drawn into a test tube containing EDTA-Na on the morning of the operation from patients who had been fasting for at least 12 hours. The plasma was separated, stored at 4°C, and used within 6 days. Total cholesterol, triglycerides, HDL, and LDL-cholesterol were determined in patients as well as in control subjects.
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Extraction of LDL
Plaque LDL was extracted by the slightly modified method of Ylä-Herttuala et al.11 Briefly, plaques were cut into small pieces, homogenized in five parts of homogenate buffer, and extracted overnight at 4°C using an orbital shaker (10 rpm, Iuchi). After centrifugation at 1000 rpm for 5 minutes, the isolated supernatant was transferred into a 4PC tube (Hitachi) and overlaid with one-third volume of homogenate buffer. The tubes were then centrifuged at 80 000 rpm for 10 minutes at 4°C (Hitachi CS120). The supernatant-discarded creamy top layer was adjusted to density of 1.18 g/mL by adding 0.5 g/mL KBr. This was followed by centrifugation at 80 000 rpm for 3.5 hours. The top layer (1 mL) and 1 mL of homogenate buffer was mixed and again centrifuged at 80 000 rpm for 2 hours. The isolated LDL fractions with a density of 1.019 to 1.090 were dialyzed at 4°C overnight against PBS (pH 7.4) containing 0.1% EDTA. The protein content of the LDL fraction was determined by using the BCA protein assay kit (Pierce). Plasma LDL isolation was by stepwise KBr density gradient ultracentrifugation; standard OxLDL was prepared by incubating LDL (0.2 mg/mL) with 5 µmol/L CuSO4 at 37°C for 3 hours as described previously.5
Determination of OxLDL Levels
By using the method described previously,5 microtiter wells precoated with DLH3 (5 µg/mL in PBS, 100 µL/well) were blocked with 1% BSA in 0.05 mol/L Tris-buffered saline, pH 8.0. In each assay, we added to the wells 100 µL of appropriately diluted plasma and plaque LDL fractions (plasma LDL, 2 to 10 µg protein/well; plaque LDL, 0.5 to 5 µg protein/well), and 0.2 to 8.0 ng/well of standard OxLDL. The plates were incubated overnight at 4°C. After washing, the remaining OxLDL was detected with 100 µL of sheep anti-human apoB IgG antibody (Boehringer) and 100 µL of alkaline phosphatase-conjugated donkey anti-sheep IgG antibody (Chemicon). The reactivity of alkaline phosphatase was measured at 405 nm after incubation for an appropriate length of time at 37°C with 100 µL substrate solution containing 1 mg/mL of p-nitrophenylphosphate hexahydrate (Wako). OxLDL per microgram of protein was calculated from the standard curve. Data from samples in the saturation range were discarded. Simultaneously, a parallel set of ELISA was run to determine the amounts of apoB in the same lipoprotein fractions by using anti-apoB mAb (OEM Concepts). For apoB measurement, 0.5 to 15 ng/well native LDL was added to each ELISA plate as the standard. The OxLDL levels were expressed as amounts of OxLDL per microgram of apoB protein.
Immunoblot Analysis
The lipoprotein fractions from carotid plaques and plasma LDL fractions were submitted to immunoblot analysis. Aliquots (1 µg protein/lane) were subjected to 3% to 12% SDS-PAGE (Bio-Rad, Japan) and electrotransferred to poly vinilidene difluoride membranes. We also used silver-stain SDS-PAGE. The membranes were blotted with anti-apoB polyclonal antibody (Binding Site Inc) and visualized with an HRP-conjugated second antibody (Chemicon) and an enhanced chemiluminescence reagent (Amersham).
Histopathological Characteristics of Plaques
Fresh plaque specimens, some of which required decalcification, were promptly fixed in methanol-Carnoy’s fluid (Ishizu) and embedded in paraffin, and 4-µm-thick sections were cut. Histopathological characteristics were examined after staining with hematoxylin and eosin and elastica van Gieson. Adjacent serial sections were stained immunohistochemically. An experienced pathologist analyzed the histological features. The level of M infiltration and the lipid core size were measured by using an image analysis program (Mac SCOPE). Intraplaque inflammation plays a key role in plaque destabilization.17 Based on the extent of M infiltration, the plaques were classified as M-poor (infiltration: <5% of total area, n=24) and M-rich (infiltration: 5% of total area, n=20). All results were cross-checked to investigate possible relationships with diverse histopathological and clinical findings (eg, the grade of angiographical stenosis [%], ultrasound echogenicity). The pathologist was not cognizant of either the plaque OxLDL levels or the patients’ clinical data.
Immunohistochemical Analysis
Antibodies used for immunohistochemical study were anti-macrophage mAb (kp-1, Dako; HAM-56, Dako), anti-
-smooth muscle actin mAb (Sigma), anti-apoB polyclonal antibody (anti-apoB; Fitzgerald), and the anti-OxPC mAb DLH3. Serial sections were deparaffinized in xylene and dehydrated in a graded series of ethanol and immunostained by using LSAB kit (Dako). They were treated overnight at 4°C with each antibody and anti-mouse IgG1 mAb (Dako) as a negative control. Some sections were treated with proteinase K (Dako) for 9 minutes at room temperature. Nuclei in all sections were stained with methylgreen.
Statistical Analysis
Data are expressed as the mean±SEM. Statistical analyses were performed on a Macintosh computer with statistical software (Stat View 5.0). The Mann-Whitney U test was used to compare the difference between two groups. The comparison among more than three groups was performed by using ANOVA followed by Scheffe’s test. Some correlations were evaluated by using the Spearman rank correlation test. Statistical significance was considered as P<0.05.
Results
Plaque LDL Is Enriched With OxLDL
The plasma OxLDL level was significantly higher in patients than in the controls (0.18±0.01 versus 0.13±0.01 ng/µg apoB, P=0.02), suggesting that OxLDL is involved in carotid artery disease, as is the case in coronary artery disease.7–9 In paired patient samples, the OxLDL level in plaque LDL fractions was 11.9±1.7 ng/µg apoB, nearly 70 times higher than in plasma (P<0.0001). The OxLDL level in LDL fractions from normal intima (1.86±0.59 ng/µg apoB) was 6 times lower than in LDL fractions from plaques (P<0.001), and the concentration of apoB in the LDL fraction recovered from normal intima was less than 40% of that of plaques. The OxLDL content in LDL fractions from normal intima was about one-tenth of that in LDL fractions from plaques when normalized by amounts of total protein (0.06±0.02 vs 0.84±0.13 ng/µg protein, P<0.0001). ApoB-containing lipoproteins in plaques correlated with plaque OxLDL levels (n=53, r=0.555, P<0.0001). These findings suggest that plaque LDL is enriched with oxidative modification.
Comparison of OxLDL Levels and Plaque Morphology
There was a strong correlation between the plaque OxLDL level and the extent of macrophage infiltration (r=0.63, P<0.0001, Figure 1). The characteristics of the M-rich and M-poor carotid atherosclerotic lesions are summarized in Table 2. The M-rich plaques were associated with plaque rupture, fibrous cap thinning, and a large-sized lipid core and correlated with ultrasonographic echolucency but not with angiographical carotid stenosis. The plasma OxLDL level in patients with M-rich plaques was significantly higher than in control plasma (0.20±0.02 vs 0.13±0.01 ng/µg apoB, P=0.02) (Figure 2A) and slightly higher than in patients with M-poor plaques (0.16±0.01 ng/µg apoB); the difference in the plasma OxLDL level between patients with M-rich- and M-poor plaques was not significant. The OxLDL level in M-rich plaques was 3.6 times higher than in M-poor plaques (19.6±2.8 vs 5.50±0.77 ng/µg apoB, P<0.0001) (Figure 2B). There was no significant difference between the OxLDL level in normal intima and M-poor plaques and between the plasma OxLDL level in the patients with M-poor plaque and the controls.
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ApoB Oxidation Analysis by Western Blot
As shown in Figure 3, Western blot analysis of LDL fractions from control and patient plasma and from carotid plaques manifested that, in the controls, native apoB had a molecular weight of 500 kDa; there was no 500-kDa apoB band in plaque LDL fractions. Smear-staining was noted in the lower molecular weight range. However, in plasma LDL from the same patient, the apoB band was almost the same as in the controls, suggesting that apoB in plaques was partially degraded. Comparison of the degree of apoB degradation in M-poor and M-rich plaques revealed a loss of native-sized apoB and the appearance of low molecular weight (up to 120 kDa) apoB in LDL from M-rich plaques. However, the changes in apoB were moderate in LDL fractions from M-poor plaques; the size of low molecular weight bands from degraded apoB was slightly larger (up to 200 kDa). Itabe et al18 reported that partially degraded apoB accumulated in lysosomes after OxLDL was taken up by cultured macrophages. The observation that apoB in M-rich plaques tended to be of smaller molecular size may be reflective of metabolic activity acting on OxLDL in macrophages present in these plaques.
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Immunohistopathology
Figure 4 shows representative cases of M-rich- and M-poor plaques, including examples of intraplaque hemorrhage (Figure 4A) and plaque rupture (Figure 4B). In M-rich, but not in M-poor plaques, accumulations of macrophage-derived foam cells and macrophages were noted (Figure 4C). The localization pattern of DLH3-antigens (Figure 4D) was consistent with that of macrophages detected by anti-macrophage antibodies (Figure 4C). DLH3-positive antigens were detected more in M-rich than M-poor plaques (Figure 4D, 4F, and 4H). This corresponds with the finding that the OxLDL level was 3.6 times higher in M-rich than M-poor plaques (Figure 2B). Foam- and macrophage-like cells showed strong staining with DLH3 (Figure 4D). The intracellular space between relatively small foam cells tended to be more strongly stained than that of giant and expanded foam cells (Figure 4F), suggesting that small foam cells are more active and that large foam cells are prone to rupture. The necrotic core was immunostained with DLH3, but the degree of staining was variable. The distribution of smooth muscle cells differed from that of DLH3 antigen. ApoB was deposited mainly in the extracellular matrix and occasionally colocalized with OxLDL in macrophage-derived foam cells.
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Comparison of OxLDL Levels, Lipid Parameters, Patient Symptoms and Risk Factors
There was no significant correlation between plasma lipid parameters and OxLDL levels in either plasma or plaques (data not shown). Plasma OxLDL was not correlated with plasma LDL (r=-0.07, P=0.63). There was also no association between plasma and plaque OxLDL levels (r=0.20, P=0.20). In symptomatic patients (n=19), the plaque OxLDL levels were higher than in asymptomatic patients (n=25), however, the difference was not significant (15.0±3.5 versus 9.6±1.4 ng/µg apoB), Furthermore, macrophage infiltration was not significantly different between symptomatic and asymptomatic patients (5.6±0.8 versus 4.4±0.6% of total area). There was also no significant difference between symptomatic and asymptomatic patients with respect to their plasma OxLDL levels. Patient risk factors appeared to have no effect on plasma and plaque OxLDL levels (data not shown).
Discussion
OxLDL is present in atherosclerotic lesions.1–4 Immunohistochemical studies using antibodies to OxLDL revealed the presence of OxLDL-related epitopes in atherosclerotic lesions.6,8,10,12 Ylä-Herttuala et al11,19 isolated the LDL fraction from human atherosclerotic lesions and compared the biological properties of OxLDL with those in normal intima. Although it is widely accepted that the atherogenic oxidation of LDL occurs predominantly in the arterial wall, to our knowledge, no quantitative analyses of OxLDL in human atherosclerotic lesions have been reported. We first determined both plasma and plaque OxLDL levels by a sandwich ELISA using DLH3 and anti-apoB antibody. Then we investigated the correlation between these levels and the morphology of atherosclerotic carotid plaques. The results showed that in carotid plaques the OxLDL level was nearly 70 times higher than in plasma from the same patient. The OxLDL level in control plasma suggests that approximately 1 in 7500 LDL particles was oxidatively modified. This is consistent with values reported previously.5 Our sandwich ELISA procedure used anti-OxPC and anti-apoB antibodies, thus facilitating the determination of preformed OxLDL in biological samples. OxLDL detected by this method can be assumed to be OxPC-apoB adduct-containing LDL particles; the results do not reflect OxLDL concentration but rather the ratio of oxidatively modified LDL particles in total LDL. Because OxPC is one of the oxidative products formed in OxLDL, it should be noted that this method might not be able to detect the total amount of OxLDL.
In the course of atherogenesis, foam cells may play a critical role in the progression of lesions. Monocyte-derived macrophages can produce cytokines, proteolytic enzymes (particular metalloproteases), and growth factors. We posit that the atherogenicity of macrophages increases with their ability to take up OxLDL and leads to plaque instability. Based on the degree of macrophage infiltration, we classified the plaques as M-rich and M-poor. The former had significantly higher rates of rupture, fibrous cap thinning, intraplaque hemorrhage, and a bigger lipid core than did M-poor plaques. This suggests that M-rich plaques tend to be unstable. Moreover, plaque OxLDL levels exhibited a strong correlation with macrophage infiltration (P<0.0001). In patients with M-rich plaques, plasma OxLDL levels were significantly higher than in the controls, suggesting that the elevation of plasma OxLDL is associated with plaque instability. Our findings support the hypothesis that the OxLDL level and the degree of macrophage infiltration determines whether atherosclerotic lesions are stable or unstable. Immunohistochemically, in M-rich plaques, macrophage infiltration was 3 times higher than in M-poor plaques and foam cells, and macrophages stained strongly for the DLH3-antigen. In M-poor plaques, on the other hand, foam cells and macrophages stained only faintly. These findings also reflect the results of direct quantitative analyses. The distribution of DLH3- and apoB-positive areas was consistent with that reported previously.6,9,20,21
Western blots using apoB antibody showed that apoB in plaques was partially degraded and that the extent of apoB degradation was related to the OxLDL level. ApoB was more extensively fragmented in M-rich (high OxLDL levels) than M-poor plaques (lower OxLDL levels). The accumulation of partially degraded apoB in lysosomes after exposure of murine macrophages to OxLDL has been reported16 and the size of degraded apoB in M-rich plaques (up to 120kDa) corresponded to the size reported for murine macrophages. We posit that the uptake and degradation of OxLDL is enhanced in M-rich plaques. It should be noted that plaque LDL would be extracted from both intracellular and extracellular spaces under our experimental conditions, because the plaques were homogenized with a polytron homogenizer. Degradation of apoB in plaque LDL was less prominent in a previous study by Ylä-Herttuala et al11 presumably because their milder extraction condition allowed them to extract extracellular LDL particles. In addition, there might be a contribution from extracellular proteases that degrade OxLDL in plaques because macrophages are an important source of extracellular proteases. Western blots detected little apoB degradation in plasma LDL fractions, presumably because the content of OxLDL in these fractions was very low. ApoB may be more susceptible to oxidative modification in plaques than in plasma; alternatively, plaques may have greater ability to retain circulation-derived OxLDL.
Smith et al22 showed that the interior of advanced human atherosclerotic lesions is a highly pro-oxidant environment containing transition metal ions. Plaque instability may induce a focal imbalance between pro-oxidants and anti-oxidant defense- and repair systems and contribute partly to high levels of plasma OxLDL. Our study did not address the origin of circulating OxLDL or the mechanisms underlying the increase in plasma OxLDL. We found no significant correlation between patient symptoms and their plasma OxLDL levels. Furthermore, the difference in the plasma OxLDL between patients with M-poor- and M-rich plaques was not significant. The timing of the CEA operation may affect the plasma OxLDL level. All of our patients had either suffered a stroke more than one month earlier or were asymptomatic at the time of the operation. We posit that the plasma OxLDL level during the chronic phase may be the stable level for the individual and that the higher plasma OxLDL level in patients with M-rich plaques may reflect the susceptibility of their LDL to oxidation. To address these issues, we are currently performing studies in which we are obtaining measurements from a large patient population in the acute stroke phase.
Ours is the first report on OxLDL levels in paired plasma and plaque samples from CEA patients with M-rich and M-poor plaques. Our results suggest that LDL undergoes further oxidation in plaques, that plasma OxLDL levels reflect oxidative conditions, and that high plasma and plaque OxLDL levels are associated with the vulnerability to rupture of carotid atherosclerotic lesions.
Acknowledgments
This study was supported by grant 12671361 for Scientific Research from the Japanese Ministry of Education. The authors are indebted to staff at Tokushima Municipal Hospital, Anan Kyoei Hospital, and Kenpo Naruto Hospital for providing carotid plaques, to Megumi Inoue for technical assistance, and to Setsuko Hase and Nao Kawasaki for secretarial assistance.
Received December 26, 2001; accepted August 4, 2002.
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S. J.A. Korporaal, M. Van Eck, J. Adelmeijer, M. Ijsseldijk, R. Out, T. Lisman, P. J. Lenting, T. J.C. Van Berkel, and J.-W. N. Akkerman Platelet Activation by Oxidized Low Density Lipoprotein Is Mediated by Cd36 and Scavenger Receptor-A Arterioscler. Thromb. Vasc. Biol., November 1, 2007; 27(11): 2476 - 2483. [Abstract] [Full Text] [PDF]
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 R. Chen, L. Yang, and T. M. McIntyre Cytotoxic Phospholipid Oxidation Products: CELL DEATH FROM MITOCHONDRIAL DAMAGE AND THE INTRINSIC CASPASE CASCADE J. Biol. Chem., August 24, 2007; 282(34): 24842 - 24850. [Abstract] [Full Text] [PDF]
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D. Segers, F. Helderman, C. Cheng, L. C.A. van Damme, D. Tempel, E. Boersma, P. W. Serruys, R. de Crom, A. F.W. van der Steen, P. Holvoet, et al. Gelatinolytic Activity in Atherosclerotic Plaques Is Highly Localized and Is Associated With Both Macrophages and Smooth Muscle Cells In Vivo Circulation, February 6, 2007; 115(5): 609 - 616. [Abstract] [Full Text] [PDF]
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 S. Tsimikas, E. S. Brilakis, R. J. Lennon, E. R. Miller, J. L. Witztum, J. P. McConnell, K. S. Kornman, and P. B. Berger Relationship of IgG and IgM autoantibodies to oxidized low density lipoprotein with coronary artery disease and cardiovascular events J. Lipid Res., February 1, 2007; 48(2): 425 - 433. [Abstract] [Full Text] [PDF]
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J. W. Gaubatz, B. K. Gillard, J. B. Massey, R. C. Hoogeveen, M. Huang, E. E. Lloyd, J. L. Raya, C.-y. Yang, and H. J. Pownall Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A2 J. Lipid Res., February 1, 2007; 48(2): 348 - 357. [Abstract] [Full Text] [PDF]
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F. D. Kolodgie, A. P. Burke, K. S. Skorija, E. Ladich, R. Kutys, A. T. Makuria, and R. Virmani Lipoprotein-Associated Phospholipase A2 Protein Expression in the Natural Progression of Human Coronary Atherosclerosis Arterioscler. Thromb. Vasc. Biol., November 1, 2006; 26(11): 2523 - 2529. [Abstract] [Full Text] [PDF]
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J. Rodenburg, M. N. Vissers, A. Wiegman, E. R. Miller, P. M. Ridker, J. L. Witztum, J. J.P. Kastelein, and S. Tsimikas Oxidized Low-Density Lipoprotein in Children With Familial Hypercholesterolemia and Unaffected Siblings: Effect of Pravastatin J. Am. Coll. Cardiol., May 2, 2006; 47(9): 1803 - 1810. [Abstract] [Full Text] [PDF]
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S. Tsimikas, J. T. Willerson, and P. M. Ridker C-reactive protein and other emerging blood biomarkers to optimize risk stratification of vulnerable patients. J. Am. Coll. Cardiol., April 18, 2006; 47(8 Suppl): C19 - C31. [Abstract] [Full Text] [PDF]
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R. P. Mason, M. F. Walter, C. A. Day, and R. F. Jacob Active Metabolite of Atorvastatin Inhibits Membrane Cholesterol Domain Formation by an Antioxidant Mechanism J. Biol. Chem., April 7, 2006; 281(14): 9337 - 9345. [Abstract] [Full Text] [PDF]
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L. Cominacini, M. Anselmi, U. Garbin, A. Fratta Pasini, C. Stranieri, M. Fusaro, C. Nava, P. Agostoni, D. Keta, P. Zardini, et al. Enhanced Plasma Levels of Oxidized Low-Density Lipoprotein Increase Circulating Nuclear Factor-Kappa B Activation in Patients With Unstable Angina J. Am. Coll. Cardiol., September 6, 2005; 46(5): 799 - 806. [Abstract] [Full Text] [PDF]
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C. Meisinger, J. Baumert, N. Khuseyinova, H. Loewel, and W. Koenig Plasma Oxidized Low-Density Lipoprotein, a Strong Predictor for Acute Coronary Heart Disease Events in Apparently Healthy, Middle-Aged Men From the General Population Circulation, August 2, 2005; 112(5): 651 - 657. [Abstract] [Full Text] [PDF]
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 S. Tsimikas, E. S. Brilakis, E. R. Miller, J. P. McConnell, R. J. Lennon, K. S. Kornman, J. L. Witztum, and P. B. Berger Oxidized Phospholipids, Lp(a) Lipoprotein, and Coronary Artery Disease N. Engl. J. Med., July 7, 2005; 353(1): 46 - 57. [Abstract] [Full Text] [PDF]
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D. M. Schrijvers, G. R.Y. De Meyer, M. M. Kockx, A. G. Herman, and W. Martinet Phagocytosis of Apoptotic Cells by Macrophages Is Impaired in Atherosclerosis Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1256 - 1261. [Abstract] [Full Text] [PDF]
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I. Goncalves, M.-L. M. Gronholdt, I. Soderberg, M. P.S. Ares, B. G. Nordestgaard, J. F. Bentzon, G. N. Fredrikson, and J. Nilsson Humoral Immune Response Against Defined Oxidized Low-Density Lipoprotein Antigens Reflects Structure and Disease Activity of Carotid Plaques Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1250 - 1255. [Abstract] [Full Text] [PDF]
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A. Zalewski and C. Macphee Role of Lipoprotein-Associated Phospholipase A2 in Atherosclerosis: Biology, Epidemiology, and Possible Therapeutic Target Arterioscler. Thromb. Vasc. Biol., May 1, 2005; 25(5): 923 - 931. [Abstract] [Full Text] [PDF]
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S. J.A. Korporaal, G. Gorter, H. J.M. van Rijn, and J.-W. N. Akkerman Effect of Oxidation on the Platelet-Activating Properties of Low-Density Lipoprotein Arterioscler. Thromb. Vasc. Biol., April 1, 2005; 25(4): 867 - 872. [Abstract] [Full Text] [PDF]
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 M E Tushuizen, M Diamant, and R J Heine Postprandial dysmetabolism and cardiovascular disease in type 2 diabetes Postgrad. Med. J., January 1, 2005; 81(951): 1 - 6. [Abstract] [Full Text] [PDF]
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M. Torzewski, P. X. Shaw, K.-R. Han, B. Shortal, K. J. Lackner, J. L. Witztum, W. Palinski, and S. Tsimikas Reduced In Vivo Aortic Uptake of Radiolabeled Oxidation-Specific Antibodies Reflects Changes in Plaque Composition Consistent With Plaque Stabilization Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2307 - 2312. [Abstract] [Full Text] [PDF]
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S. Tsimikas, J. L. Witztum, E. R. Miller, W. J. Sasiela, M. Szarek, A. G. Olsson, G. G. Schwartz, and for the Myocardial Ischemia Reduction with Aggress High-Dose Atorvastatin Reduces Total Plasma Levels of Oxidized Phospholipids and Immune Complexes Present on Apolipoprotein B-100 in Patients With Acute Coronary Syndromes in the MIRACL Trial Circulation, September 14, 2004; 110(11): 1406 - 1412. [Abstract] [Full Text] [PDF]
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M.-L. Liu, K. Ylitalo, R. Salonen, J. T. Salonen, and M.-R. Taskinen Circulating Oxidized Low-Density Lipoprotein and Its Association With Carotid Intima-Media Thickness in Asymptomatic Members of Familial Combined Hyperlipidemia Families Arterioscler. Thromb. Vasc. Biol., August 1, 2004; 24(8): 1492 - 1497. [Abstract] [Full Text] [PDF]
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S. Tsimikas, H. K. Lau, K.-R. Han, B. Shortal, E. R. Miller, A. Segev, L. K. Curtiss, J. L. Witztum, and B. H. Strauss Percutaneous Coronary Intervention Results in Acute Increases in Oxidized Phospholipids and Lipoprotein(a): Short-Term and Long-Term Immunologic Responses to Oxidized Low-Density Lipoprotein Circulation, June 29, 2004; 109(25): 3164 - 3170. [Abstract] [Full Text] [PDF]
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 B Gonzalez-Timon, M Gonzalez-Munoz, C Zaragoza, S Lamas, and E.M Melian Native and oxidized low density lipoproteins oppositely modulate the effects of insulin-like growth factor I on VSMC Cardiovasc Res, February 1, 2004; 61(2): 247 - 255. [Abstract] [Full Text] [PDF]
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G. P. Rossi, M. Cesari, R. De Toni, M. Zanchetta, G. Maiolino, L. Pedon, C. Ganzaroli, P. Maiolino, and A. C. Pessina Antibodies to Oxidized Low-Density Lipoproteins and Angiographically Assessed Coronary Artery Disease in White Patients Circulation, November 18, 2003; 108(20): 2467 - 2472. [Abstract] [Full Text] [PDF]
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 M. Artwohl, W. F. Graier, M. Roden, M. Bischof, A. Freudenthaler, W. Waldhausl, and S. M. Baumgartner-Parzer Diabetic LDL Triggers Apoptosis in Vascular Endothelial Cells Diabetes, May 1, 2003; 52(5): 1240 - 1247. [Abstract] [Full Text] [PDF]
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 M Uno, K T Kitazato, K Nishi, H Itabe, and S Nagahiro Raised plasma oxidised LDL in acute cerebral infarction J. Neurol. Neurosurg. Psychiatry, March 1, 2003; 74(3): 312 - 316. [Abstract] [Full Text] [PDF]
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