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Journal of Lipid Research, Vol. 47, 2208-2214, October 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology
* Department of Internal Medicine, Medical School, University of Ioannina, 45110 Ioannina, Greece
Laboratory of Physiology, Medical School, University of Ioannina, 45110 Ioannina, Greece
Laboratory of Biochemistry, Department of Chemistry, Medical School, University of Ioannina, 45110 Ioannina, Greece
** Department of Radiology, Medical School, University of Ioannina, 45110 Ioannina, Greece
Published, JLR Papers in Press, August 3, 2006.
1 To whom correspondence should be addressed. e-mail: atselep{at}uoi.gr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary key words antibody • oxidized low density lipoprotein • platelet-activating factor acetylhydrolase • phospholipase A2
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Oxidation of LDL in vitro occurs in three phases: i) the lag phase, during which consumption of the endogenous antioxidants occurs; ii) the propagation phase, in which the sn-2 unsaturated fatty acids of phosphatidylcholine molecules are rapidly oxidized to their hydroperoxides; and iii) the decomposition phase, when these hydroperoxides are cleaved, generating reactive aldehydes, such as malondialdehyde and 4-hydroxynonenal, as well as oxidized phospholipids (OxPL) containing short acyl groups at the sn-2 position (4). OxPL can be hydrolyzed into lysophosphatidylcholine (lyso-PC) by the LDL-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor (PAF) acetylhydrolase (5). Lp-PLA2 exhibits a Ca2+-independent phospholipase A2 activity and preferentially hydrolyzes phospholipids containing short acyl groups at the sn-2 position, such as PAF and OxPL (5).
OxLDL is immunogenic, and its constituents OxPL, aldehydes, and lyso-PC play important roles in OxLDL antigenicity, participating in the formation of several epitopes (6, 7). These epitopes are recognized by specific autoantibodies, which are present in serum of healthy individuals as well as in various disease states, including atherosclerotic diseases (reviewed in 8). Because the extent of LDL oxidation and the LDL-associated Lp-PLA2 activity significantly influence the levels of OxPL, lyso-PC, and aldehydes during LDL oxidation (9), these factors could also influence the types of epitopes formed on OxLDL and consequently the specificity and the plasma levels of autoantibodies against OxLDL. Indeed, we recently showed that the extent of LDL oxidation and the activity of LDL-associated Lp-PLA2 significantly affect the antibody titers against OxLDL (10).
Tendon xanthomas are deposits of lipid and connective tissue in tendons and are commonly found in patients with severe hyperlipidemia, such as familial hypercholesterolemia (FH) (11–13). Importantly, it has been shown that the presence of tendon xanthomas in patients with FH is associated with increased cardiovascular risk independently of the underlying LDL receptor gene mutation (14). The composition of tendon xanthomas exhibits many similarities to that of atherosclerotic plaques (13). Indeed, tendon xanthomas are characterized by the extracellular deposition of unesterified and esterified cholesterol as well as connective tissue and by the accumulation of foam cells (15, 16). The underlying mechanisms by which some FH patients develop tendon xanthomas and others do not remain to be established. Only recently, it was demonstrated that macrophages from FH patients with tendon xanthomas are more sensitive to the actions of OxLDL compared with cells from FH patients without tendon xanthomas (17).
Autoantibodies against OxLDL may play important roles in atherosclerosis (8, 18). To the best of our knowledge, there is a paucity of data concerning the autoantibody titers in FH patients with respect to the presence of tendon xanthomas. Therefore, the aim of this study was to evaluate the autoantibody titers against various types of mildly OxLDL as well as the plasma levels of OxLDL and Lp-PLA2 activity in relation to Achilles tendon thickness (ATT) determined by sonography in heterozygous FH patients.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1.5 times the upper limit of normal), renal insufficiency (serum creatinine > 1.6 mg/dl), proteinuria (>0.5 g/day), diabetes mellitus (fasting plasma glucose concentration 126 mg/dl or use of antidiabetic medications), hypertension (arterial blood pressure > 140/90 mmHg or use of antihypertensive medications), and thyroid-stimulating hormone levels > 5 µU/ml. None of the participants was taking lipid-lowering drugs, including statins, or any other medication known to affect lipid metabolism, including hormonal therapy, during the last 24 weeks. During the study, all patients were on an isocaloric diet, and no significant change in body weight for at least 3 months before entry into the study was reported. Blood samples were obtained after a 12 h overnight fast. Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of the University Hospital of Ioannina.
Ultrasound scanning procedure
All examinations were performed using a high-resolution B-mode ultrasound machine (Accuson XP 128) and a linear array 7 MHz transducer on the same day as the blood collection. The subjects were examined in the prone position with the foot hanging free over the end of the table. All measurements were made on saggital scans. The ATT was measured bilaterally, 4 cm above the insertion site to tuber calcanei. Special care was taken to place the transducer perpendicular to the tendon, avoiding an artificially hypoechogenic pattern. A supplementary examination was performed on transverse scans to reveal the presence of xanthomas outside the point of measurements. The same radiologist performed all examinations without knowing the clinical status of the subject. The echogenicity of Achilles tendon was classified into three grades: grade 1, normal (fibrillar structure of the tendon preserved); grade 2, diffuse (heterogeneous echo pattern); and grade 3, xanthomas (focal hypoechoic lesions) (13).
Analytical methods
All laboratory determinations were performed after patients had fasted for 12 h overnight. Serum levels of total cholesterol, HDL-cholesterol, and triglycerides were determined enzymatically on the Olympus AU600 Clinical Chemistry analyzer (Olympus Diagnostica, Hamburg, Germany). Serum LDL-cholesterol was calculated using the Friedewald formula (provided that triglycerides levels were <350 mg/dl) (21). ApoB-100 levels were measured with a Behring Nephelometer BN100 (Liederbach, Germany) (22).
Preparation of various types of OxLDL
LDL (d = 1.019–1.063 g/ml) was isolated by sequential ultracentrifugation from pooled fresh plasma (23). Before oxidation, a part of purified LDL was incubated with 0.5 mM pefabloc for 1 h at 37°C to irreversibly inactivate the Lp-PLA2; then it was dialyzed extensively against 10 mM PBS to remove the excess of pefabloc (10). LDL with active Lp-PLA2 or pefabloc-treated LDL [containing inactive Lp-PLA2 and denoted LDL(–)], at a final concentration of 100 µg protein/ml, was oxidized in the presence of 5 µM CuSO4 for 3 h at 37°C. The kinetics of oxidation were determined by monitoring the increase in the absorbance at 234 nm every 10 min (10). Oxidation of LDL or LDL(–) was terminated by the addition of 0.01% (v/v) EDTA at the end of the lag phase [OxLDLL or OxLDL(–)L, respectively)], at the end of the propagation phase [OxLDLP or OxLDL(–)P, respectively], or at 3 h of oxidation during the decomposition phase [OxLDLD or OxLDL(–)D, respectively]. The cutoff point between the lag and propagation phases was determined as described recently (10). All types of OxLDL were stored at 4°C under nitrogen for up to 4 weeks.
Measurement of antibody titers
The serum autoantibody titers of IgG class against all forms of OxLDL were determined by an ELISA method, as we described recently (10). Briefly, ELISA microplates were coated with 10 µg of protein of each type of OxLDL described above or native LDL and incubated overnight at 4°C. After washing, the microplates were postcoated with 1% gelatin. After an extensive wash, 50 µl of the serum samples (diluted 1:50, v/v) was added to each well and incubated for 1 h at room temperature. To estimate the nonspecific binding of antibodies, experiments were also performed in gelatin-coated wells. After incubation, microplates were washed and then 50 µl of an HRP-conjugated rabbit anti-human IgG monoclonal antibody (Dako Cytomation) (diluted 1:1,000, v/v) was added to the wells and incubated for 1 h at room temperature. After the removal of the unbound HRP-conjugated secondary antibody, enzyme reaction was performed using o-phenylenediamine and H2O2 as substrates, and the optical density was measured at 492 nm (10). The serum autoantibody titers of IgM class against all forms of OxLDL were also determined by the same method using 50 µl of the serum samples (diluted 1:10, v/v) and HRP-conjugated rabbit anti-human IgM monoclonal antibody (Dako Cytomation) (diluted 1:2,000, v/v). The results were expressed as the ratio of antibody binding to various types of OxLDL or OxLDL(–) versus native LDL or LDL(–), respectively. It should be noted that nonspecific binding (the binding of serum samples to gelatin-coated wells) was remarkably low, at 0.021 ± 0.005 and 0.034 ± 0.008 for IgG and IgM antibodies, respectively.
Measurement of OxLDL levels
Plasma levels of OxLDL were measured by a competitive enzyme-linked immunosorbent assay using a specific murine monoclonar antibody (4E6) according to the instructions provided by the manufacturer (Mersodia, Uppsala, Sweden). The specificity of this method was studied by performing the assay in five different plasma samples in which 5 or 15 ng of protein of native LDL or OxLDLD was added exogenously. Intra-assay and interassay coefficients of variation of the assay were 6.0% and 7.0%, respectively.
Determination of Lp-PLA2 activity
Lp-PLA2 activity in plasma and in HDL-rich plasma (HDL-Lp-PLA2 activity) was measured by the trichloroacetic acid precipitation procedure using 1-O-hexadecyl-2-[3H-acetyl]sn-glycero-3-phosphocholine ([3H]PAF) as a substrate (100 µM final concentration), as we described previously (22). The reaction was performed for 10 min at 37°C, and Lp-PLA2 activity was expressed as nmol PAF degraded/min/ml plasma.
Statistical analysis
All results are expressed as means ± SD. Continuous variables were tested for lack of normality by the Kolmogorov-Smirnov test. For comparisons between individual groups, statistical analysis was performed by the independent-samples t-test for parameters with normal distribution; otherwise, the Mann-Whitney U-test was used. To assess the correlations between the various parameters, Pearson and Spearman correlation coefficients were calculated. To compare antibody titers between the different phases of oxidation, we also used one-way ANOVA. Moreover, stepwise multiple regression analysis was performed to examine factors independently associated with ATT in FH patients. Thus, in this model, we used ATT as a dependent variable and all parameters that were significantly correlated with ATT in the univariate analysis as independent variables. All statistical analyses were carried out with SPSS 12.0 (SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Autoantibody titers against all types of OxLDL
We determined the autoantibody titers of IgG and IgM class against three different types of OxLDL (OxLDLL, OxLDLP, and OxLDLD) and three types of OxLDL(–) [OxLDL(–)L, OxLDL(–)P, and OxLDL(–)D] prepared as described in Methods.
IgG and IgM antibody titers against all types of OxLDL and OxLDL(–) were detected in FH patients as well as in controls. The IgG titers in FH patients were significantly higher when the OxLDLP and OxLDLD types were used as antigens compared with OxLDLL, the titers against OxLDLD being higher than those against OxLDLP (Fig. 1 ). By contrast, FH patients exhibited higher IgM titers against OxLDLL compared with OxLDLP and OxLDLD types, the titers against OxLDLD being lower than those against OxLDLP. The same phenomenon was observed when the OxLDL(–) types were used as antigens (Fig. 1). Furthermore, the IgG titers against each type of OxLDL(–) were higher compared with the respective type of OxLDL, whereas the opposite phenomenon was observed for the IgM titers (Fig. 1). Similar results were obtained for the IgG or IgM titers measured in the control population (data not shown). Interestingly, FH patients exhibited significantly higher IgG autoantibody titers against OxLDLP and OxLDLD compared with controls (Table 2 ), whereas no difference in the IgM titers against any type of OxLDL or OxLDL(–) was observed between the two groups (data not shown). It should be noted that no correlation was found between the IgG or IgM titers against any type of OxLDL and the plasma levels of OxLDL.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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OxLDL is present in human plasma, and several studies have been devoted to detecting OxLDL by ELISA methods using monoclonal antibodies that recognize primarily a specific type of OxLDL or a specific epitope on OxLDL (21, 24–26). One of these methods uses the monoclonal antibody 4E6, which is directed against a conformational epitope generated as a consequence of the substitution of at least 60 lysine residues of apoB-100 with aldehydes (27). Using this assay, we show that FH patients exhibit higher OxLDL plasma levels as well as a higher ratio of OxLDL to apoB-100 levels compared with controls, results that are in accordance with recently published data (28). OxLDL was positively correlated with LDL-cholesterol and apoB-100, suggesting that LDL is an important determinant of the plasma levels of OxLDL as determined with the present assay. However, using different antibodies against OxLDL, other studies failed to show any correlation between OxLDL and LDL levels (25, 29). In this regard, we show that the present assay does not have a high specificity for OxLDL and that it also detects nonoxidized LDL in plasma. However, the ratio of OxLDL to apoB-100 in FH patients is 2-fold higher compared with that in controls, a phenomenon not observed for the ratio of LDL-cholesterol to apoB-100, suggesting that although this method does not have a high specificity for OxLDL, it may give useful information for the OxLDL levels in plasma when they are expressed as a ratio to apoB-100 levels. It is important that further studies be performed before a conclusion is drawn regarding the specificity of the methods used to detect OxLDL in plasma.
This study shows for the first time that OxLDL is positively correlated with total plasma Lp-PLA2 activity. This correlation could be simply attributed to the fact that plasma Lp-PLA2 is associated mainly with LDL, which is positively correlated with OxLDL. However, enzyme activity was also positively correlated with the ratio of OxLDL to apoB-100; thus, we may not exclude the possibility that the increased Lp-PLA2 activity in FH patients leads to the liberation of increased amounts of free aldehydes (through the degradation of OxPL). These react with lysine residues of apoB-100 (30), generating increased amounts of immunogenic epitopes on OxLDL recognized by 4E6. In contrast to total plasma Lp-PLA2, HDL-Lp-PLA2 activity is negatively correlated with plasma OxLDL levels. This phenomenon could be attributed to the HDL-mediated protection of LDL from oxidation, in which HDL-Lp-PLA2 plays an important role (5).
Despite the increased plasma levels of OxLDL in FH patients, this parameter is not correlated with ATT. Thus, although OxLDL is important for lipid deposition and the formation of foam cells in Achilles tendons (15), its plasma levels may not be a determining factor for the formation of tendon xanthomas. Indeed, macrophages from FH patients with tendon xanthomas exhibit higher predisposition to foam cell formation in the presence of OxLDL and are more sensitive to the inflammatory activities of OxLDL compared with macrophages from FH patients without tendon xanthomas, suggesting that the important determinant for their formation is the genetically determined higher sensitivity of macrophages to OxLDL actions (17).
This study further showed that the autoantibody titers of IgG class against OxLDLP and OxLDLD are increased in FH patients compared with controls, whereas no difference between these two groups was observed in the IgM titers against all types of OxLDL or OxLDL(–). Contrasting results have been published regarding whether the IgG antibodies against OxLDL have any causal role in atherosclerotic diseases (8, 21, 31–33), whereas most of the studies suggest that IgM OxLDL antibodies may play an antiatherogenic role (reviewed in 18). In this regard, lower plasma IgM OxLDL autoantibodies have been noted in patients with myocardial infarction (21, 30), stable coronary artery disease (30, 34), and borderline hypertension (33) and have been inversely associated with increased carotid intima thickness (34). Because only the IgG titers against OxLDLP and OxLDLD are increased in our FH patients, we suggest that IgG antibodies against mildly oxidized forms of LDL (up to 3 h of oxidation) may be primarily involved in the premature atherosclerosis observed in FH. By contrast, a previous study did not find any difference in the autoantibody titers between heterozygous FH patients and normolipidemic controls; however, extensively OxLDL (at least 12 h of oxidation) or malondialdehyde-LDL were used as antigens in that study (32). Overall, we suggest that the extent of LDL oxidation significantly influences the plasma autoantibody levels in FH patients. This is further supported by our recently published data in patients with stable coronary artery disease (10). According to these results, the OxLDLP and OxLDLD types are enriched in lyso-PC and free aldehydes but contain significantly less OxPL compared with the corresponding OxLDL(–) types, as a result of the Lp-PLA2-mediated hydrolysis of OxPL. Thus, we suggest that epitopes formed in the presence of lyso-PC and aldehydes may be primarily responsible for the increased IgG titers observed in FH patients. However, these epitopes need to be characterized in future studies.
Importantly, only the IgG titers against OxLDLD are independently associated with ATT, suggesting that these antibodies are involved in the pathogenesis of Achilles tendon xanthomas. The pathogenesis of tendon xanthomas resembles that of atherosclerotic plaque (13). The role of immune mechanisms in atherosclerosis is complicated, and there are contradictory results regarding whether autoantibodies against OxLDL or OxLDL immune complexes may promote or inhibit atherosclerosis (8, 18). Thus, it remains to be established whether the IgG antibodies against OxLDLD promote or inhibit the development of Achilles tendon xanthomas. Furthermore, prospective studies are required to demonstrate whether the IgG titers against OxLDLD in FH patients may have any prognostic value for the development of Achilles tendon xanthomas, which are associated with cardiovascular risk factors and a higher incidence of coronary artery disease in this population (14).
In conclusion, this study demonstrates for the first time that IgG titers against OxLDLD are independently associated with ATT, suggesting that they may play a role in the pathogenesis of ATX in heterozygous FH patients.
Manuscript received March 6, 2006 and in revised form July 19, 2006.
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