Department of Pathophysiology Medical School, National University of Athens,
1 Clinical and Molecular Epidemiology Unit, Department of Hygiene and Epidemiology, University of Ioannina, School of Medicine, Ioannina and
2 Department of Radiology, Onassis Cardiac Surgery Center, Athens, Greece
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Abstract |
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Methods. We evaluated premenopausal women with APS in comparison with age-matched groups of patients with SLE [positive or negative for anticardiolipin (aCL) antibodies] or rheumatoid arthritis (RA), and healthy subjects. Thirty-three subjects in each group were assessed for cardiovascular risk factors, including a detailed lipid profile. Ultrasonography of carotid and femoral arteries assessed the intima-media thickness (IMT) and the presence of atherosclerotic plaque.
Results. Atherosclerotic plaques were detected in 5, 2, 4, 1 and 1 subject in the five groups respectively. APS patients had significantly more affected vessels than RA patients and healthy controls (P=0.042 and P=0.016, respectively), but not compared with SLE patients. No consistent differences in IMT, traditional cardiovascular risk factors or lipid parameters were detected among the five groups. The odds for atherosclerosis independently increased 1.19-fold per year of increasing age [95% confidence interval (CI) 1.081.31; P=0.001), 1.019-fold per 1 mg/dl increase in low-density lipoprotein (LDL) (95% CI 1.0031.036; P=0.020), 1.035-fold per additional 1 g of methylprednisolone equivalent cumulative corticosteroid dose (95% CI, 0.9961.074; P=0.074), and 4.35-fold in the presence of APS or SLE (95% CI 0.7525.2; P=0.10). Neither aCL nor anti-ß2GPI antibodies were associated with atherosclerosis.
Conclusion. Premenopausal APS and SLE women have an increased prevalence of carotid and femoral plaque that is not accounted for by other predictors of atherosclerosis, including age, lipid parameters and cumulative steroid dose.
KEY WORDS: Antiphospholipid syndrome, Systemic lupus erythematosus, Atherosclerosis, Vascular ultrasound, Intima-media thickness, Carotid artery, Femoral artery.
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Introduction |
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A suitable disease model to study the association of autoimmunity with atherosclerosis is the acquired, immune-mediated thrombophilia known as antiphospholipid syndrome (APS), defined as a combination of thrombosis or pregnancy morbidity with aCL antibodies or lupus anticoagulant (LA) [9]. aCL antibodies occurring in the context of APS often recognize the ß2-glycoprotein I (ß2GPI) [10]. ß2GPI bound to oxLDL can be recognized by aCL antibodies, thus increasing lipoprotein uptake by macrophages [11]. Furthermore, immunolocalization of ß2GPI to human atherosclerosis plaques [12] suggests that anti-ß2GPI antibodies may be implicated in atheroma progression.
Despite experimental work in animals, there are inconclusive data associating atherosclerosis with APS or with aCL or anti-ß2GPI antibodies in humans [1317]. Prior studies have been cross-sectional and have typically included one-time antibody measurements without control groups. Thus, we undertook a controlled study to detect atherosclerotic lesions in patients with APS, and appropriate control groups, including SLE patients, either positive or negative for aCL antibodies, patients with rheumatoid arthritis (RA) and healthy individuals, were also evaluated.
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Patients and methods |
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Detection of antibodies
Antinuclear antibodies were detected by immunofluorescence, using Hep-2 cells as substrate [20]. Anti-dsDNA, aCL and anti-ß2GPI antibodies were detected by ELISA (enzyme-linked immunosorbent assay) [2123]. aCL and anti-ß2GPI positivity was defined by serum reactivity 5 standard deviations higher than that of the mean of normal subjects at least on two occasions, 6 weeks apart. LA was assayed using the activated partial thromboplastin time, kaolin clotting time and dilute viper venom time [24].
Each subject was evaluated for the presence of aCL antibodies at least twice. SLE and APS patients had been evaluated a median of five times, and RA patients and healthy controls two times. Individuals with one positive and one negative aCL measurement were excluded. For two patients, APS was defined on the basis of the presence of LA rather than aCL. Exclusion of these two patients yielded similar results (not shown).
Measurement of lipids and lipoproteins
Total lipids, total cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), triglycerides, apolipoproteins (Apo) A1 and B, and lipoprotein [Lp(
)] were measured by standardized laboratory tests, using automatic analysers (Hitachi-911 and Hitachi 917; Roche Diagnostics, Mannheim, Germany). Lipoprotein electrophoresis was performed on an agarose gel using the rapid electrophoresis (REP) system (Helena's Laboratories, Gateshead, UK).
Detection of atherosclerosis
Ultrasonography of both carotid and femoral arteries was performed with a 105 MHz linear array transducer with ATL Ultra Mark 9 High Definition Imaging (ATL, Minneapolis, MN, USA), as described [25, 26]. We collected information on intima-media thickness (IMT) of the far (inner) wall and the presence or absence of atherosclerotic plaques. On a longitudinal two-dimensional image of the artery, the wall is displayed as two bright white lines separated by a hypoechogenic space. The distance between the leading edge of the first bright line and the leading edge of the second bright line indicates the IMT [27]. The actual measurements of IMT were performed off-line, using frozen images from videotape. Each measurement reflects the median of three frozen images obtained by three measurements of each area. The coefficient of variation was 10.4%. The arteries were also evaluated off-line for the presence of atherosclerotic lesions on the near or the far wall. Plaques were defined as focal widening relative to adjacent segments, with protrusion into the lumen of calcified or non-calcified material.
Statistical analysis
The main contrast was between APS and healthy subjects and the study had 80% power (=0.05) to detect a difference of 20% between the two groups, assuming that the prevalence of atherosclerotic lesions in the carotids or femoral arteries in healthy premenopausal women would be approximately 3%. The study had 80% power to detect smaller differences (in the range of 10%) in the contrast between the APS and SLE groups combined vs the other controls. ß2GPI comparisons between groups used Fisher's exact test for 2x2 and 2xn tables (discrete data) and KruskalWallis analysis of variance for continuous data. For analyses involving data with measurements from two sites per individual (right and left blood vessels), a generalized linear model was used with a type III sum-of-squares design. Group membership and the side examined were used as fixed effects variables. Random effects modelling yielded similar results. We also considered models adjusted for body mass index (BMI).
We also used univariate and multivariate logistic regressions to examine relationships between various candidate predictors and the presence of arterial lesions. Multivariate models built with forward or backward selection of variables according to likelihood ratio criteria usually yielded similar results. The logistic regressions were conducted in the total study population, and separate analyses were also conducted limited to the three groups of patients with APS or SLE. Age, BMI, cumulative dose of steroids, lipid parameters, group membership and cardiovascular risk factors were considered as predictors in both analyses. aCL and anti-ß2GPI antibodies and duration of disease were also considered as candidate predictors in analyses limited to the three APS or SLE groups. All the reported P values are not adjusted for multiple comparisons.
Analyses were conducted in SPSS 10.0 (SPSS Inc., Chicago, IL, USA) and StatXact 3.0 (Cytel, Boston, MA, USA). P values are two-tailed.
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Results |
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The median (interquartile range) titre for immunoglobulin (Ig) G aCL antibodies was 150 (100230) for APS subjects, 167 (111264) for SLE/aCL-positive subjects, 50 (1382) for SLE/aCL-negative subjects and 50 (3450) for RA subjects. The titres for IgM aCL were 154 (1311246), 88 (41213), 50 (1465) and 50 (4673) respectively. The titres for anti-ß2GPI antibodies were 174 (138422), 67 (5182), 59 (4095) and 26 (2630), and for their IgM counterparts 59 (41203), 152 (102230), 53 (5080) and 70 (53140). Median titres for healthy controls were very low (1360) for all these antibodies.
Prevalence of atherosclerotic disease in the carotid and femoral arteries
Table 2 shows the prevalence of detected atherosclerotic vascular disease in either the carotid or femoral arteries. The number of subjects with documented vascular disease was higher in the APS group {5/33 [15%, 95% binomial confidence interval (CI), 532%]} than in any other group. However, the difference between the groups was not formally statistically significant (P=0.33). The difference between APS and healthy controls was not formally significant (P=0.20). The difference between the combined APS and SLE groups vs the other controls was of borderline significance (P=0.078).
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The five groups differed significantly among themselves (P=0.042) in the total number of affected vessels. Patients with either APS or SLE (regardless of whether they had aCL antibodies) had a higher prevalence of detectable lesions compared with patients with RA and with healthy controls (P=0.003). In pair-wise comparisons, the APS group differed significantly from the RA and healthy control groups (P=0.042 and P=0.016 respectively).
Of the 13 subjects with documented vascular disease of the carotid and/or femoral arteries, 1 had diabetes mellitus, 1 had CAD, 1 had a BMI over 33 kg/m2, 5 had a history of at least 15 pack-years of smoking, 2 had hypertension and 3 had taken cumulatively at least the equivalent of 30 g of methylprednisolone, and none was using oral contraceptives. In all, only 6 subjects without any of these risk factors had documented vascular disease [2/25, 1/24, 2/23, 0/21 and 1/23 in the APS, SLE/aCL-positive, SLE/aCL-negative, RA and healthy control groups respectively (P=0.86)].
There was no evidence of any significant difference in the IMT between the five groups. Mean (S.D.) carotid IMT was 0.53 (0.14), 0.54 (0.12), 0.54 (0.12), 0.56 (0.12) and 0.56 (0.11) mm in the five groups respectively, and corresponding femoral IMT values were 0.53 (0.16), 0.52 (0.10), 0.55 (0.23), 0.52 (0.13) and 0.51 (0.11) mm (between-groups P=0.58 for carotid IMT; P=0.69 for femoral IMT by fixed effects). Results were similar with random effects modelling and were not altered by adjustment for BMI (not shown).
Lipid profile
The five compared groups did not differ significantly in the levels of total lipids, total cholesterol and LDL. There was a trend for differences in HDL level, with a lower level in the APS group compared with other groups (Table 3). Modest differences were seen in the triglyceride levels, with higher mean levels in the APS and SLE/aCL-negative groups. Conversely, Lp(
) levels tended to be higher in the SLE/aCL-positive and RA groups. There was no consistent pattern in these differences. None of the P values were formally significant after adjustment for the number of comparisons. There were no between-group differences in the levels of ApoA1 and ApoB.
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Predictors of atherosclerotic lesions
Analyses including all 165 subjects showed that age, total cholesterol, LDL and the cumulative dose of corticosteroids were associated with the occurrence of carotid or femoral lesions in univariate analyses, while a strong, yet statistically borderline predisposing effect was also seen for patients with APS or SLE (Table 4). In multivariate modelling, age, LDL, cumulative corticosteroids and SLE or APS were independent predictors of arterial lesions. Multivariate models should be interpreted cautiously, given the limited number of observations. The odds increased 1.19-fold per year of increasing age (95% CI 1.081.31; P=0.001), 1.019-fold per each 1 mg/dl increase in LDL (95% CI 1.0031.036; P=0.020), 1.035-fold (95% CI 0.9961.074; P=0.074) for each additional 1 g of methylprednisolone equivalent, and 4.35-fold (95% CI 0.7525.2; P=0.10) in the presence of APS or SLE.
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Discussion |
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A larger IMT and the presence of plaque of carotid or femoral arteries [28] are complementary but distinct risk factors for CAD events [29]. Thus, it is possible that only one of these two parameters may be affected in APS and/or SLE. IMT measurements are strong prognosticators of CAD risk [30, 31]. Our data suggest that there are probably no large differences in IMT between the study groups, although modest differences could have been missed due to noise from measurement error. Other investigators have observed that among patients with juvenile-onset systemic lupus erythematosus, the IMT was significantly higher (but only by 0.03 mm on average) compared with healthy age-matched controls [32]. However, in another controlled study [33] of 22 SLE women (including only four with APS) and 44 controls (mean age 41 yr), no differences were observed in IMT values despite a significantly higher prevalence of plaque in SLE patients (41 vs 9%). Discrepancies could be due to the different age range of the subjects or to random error.
Our study was not designed to examine subgroups within the APS group. There was no strong suggestion that atherosclerosis was detected specifically in patients with a history of arterial or CNS disease, but subgroup differences cannot be excluded. As there is strong clustering of events in the arterial or venous circulation among APS patients [34], it is possible that atherogenesis may be implicated directly in arterial clinical manifestations of selected patients.
Studies associating atherosclerosis with aCL or anti-ß2GPI antibodies in humans have mostly been cross-sectional. Lecerf et al. [13] described six APS patients with atherosclerosis, but five of them were older than 63 yr. Shortell et al. [14] identified 19 patients with APS among 1078 individuals with atherosclerosis. Nityanand et al. [15] detected aCL antibodies in 14.5% of young individuals with atherosclerosis, while Romero et al. [16] associated the presence of anti-ß2GPI antibodies with premature atherosclerosis in an SLE patient cohort, including patients with secondary APS. Limaye et al. [17], in a study of patients having undergone angioplasty for CAD reported a prevalence of 7.6% for aCL or anti-ß2GPI antibodies.
Known powerful predictive factors for atherosclerosis, such as age, sex, corticosteroid use and the lipid profile, must be taken into account in order to identify additional predictive factors for atherosclerosis. An uncontrolled study of 175 women with SLE of a higher mean age (45 yr) found that independent predictors of carotid plaque included age, hypertension, higher levels of LDL and prolonged treatment with steroids [35]. These factors proved to be equally important in our younger study cohort. After adjusting for the above factors, we observed that SLE and APS, but not aCL or anti-ß2GPI antibodies, still conferred an increased risk of atherosclerosis. Lack of control groups and residual confounding in earlier studies may have yielded a spurious impression of strong associations between aCL or anti-ß2GPI and atherosclerosis. Alternatively, measurement errors due to single antibody determinations may also be responsible for spurious associations. Finally, the age range of the study population may be influential. Overall, our study counters the hypothesis that high titres of aCL and anti-ß2GPI antibodies are directly related to an atherogenic process in premenopausal patients with SLE and APS. Nevertheless, both APS and SLE seem to confer an increased risk for plaque above and beyond what could be explained by their effect on known cardiovascular risk factors.
The reason for this additional risk is unclear. Crude titres of aCL and anti-ß2GPI antibodies may not be representative of the functional effects of these antibodies. Only high-avidity aCL or anti-ß2GPI antibodies may be associated with pathogenic consequences [23]. Other autoimmune processes may be involved in atherosclerosis. Or other inflammatory parameters, not necessarily reflecting autoimmunity, may underlie the excess atherosclerosis risk. For example, inflammatory markers such as C-reactive protein and fibrinogen have been related to atherosclerotic lesions in SLE patients [35]. C-reactive protein in particular has been emerging as a strong predictor of CAD outcomes [36, 37] and has also been linked to the morphology of plaque lesions [38].
The clinical consequences of the increased risk of atherosclerosis in APS and SLE could be considerable. While the rate of documented plaque lesions was modest in our study, it is still worrying when we consider that it pertains to patients who were mostly in their thirties. As other disease manifestations are better controlled with current therapeutic modalities, the relative importance of atherosclerotic disease may increase further. More meticulous screening for cardiovascular risk factors and more aggressive management of the identified risk factors may thus need to be considered for both APS and SLE patients.
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Acknowledgments |
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Notes |
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References |
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