Abstract
Objective. Patients with systemic lupus erythematosus (SLE) have excess cardiovascular morbidity and mortality due to accelerated atherosclerosis that cannot be attributed to traditional cardiovascular risk factors alone. Variant alleles of the mannose-binding lectin gene (MBL2) causing low serum concentrations of functional mannose-binding lectin (MBL) are associated with SLE and development of atherosclerosis. Recent studies show that these variant alleles are associated with increased risk of arterial thrombosis and cardiovascular disease in patients with SLE. Intima-media thickness of the common carotid artery (ccIMT) is a validated noninvasive anatomic measure of subclinical atherosclerosis. In a cross-sectional study we examined the relation among ccIMT, MBL2 genotypes, and serum concentrations of MBL.
Methods. The MBL2 extended genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, YO/YO) and serum concentrations of MBL were determined in 41 outpatients with SLE. ccIMT was measured by means of ultrasonography. Traditional and nontraditional cardiovascular risk modifiers were assessed and controlled for.
Results. Using nonparametric Mann-Whitney tests we found a significant difference in ccIMT between low-expressing (XA/XA+YA/YO+XA/YO+YO/YO) and high-expressing (YA/YA+YA/XA) MBL2 genotypes (p = 0.034). The difference in ccIMT remained significant in multivariable analysis adjusting for traditional and nontraditional cardiovascular risk modifiers (p = 0.049). ccIMT was negatively correlated to serum concentrations of MBL (Spearman rho = −0.33, p = 0.037). This relation also remained significant in multivariable analysis (p = 0.042).
Conclusion. In this group of SLE patients, MBL2 low-expressing genotypes and low serum levels of MBL were correlated with ccIMT, independent of the effects of traditional and nontraditional cardiovascular risk modifiers.
Patients with systemic lupus erythematosus (SLE) have increased incidence of preclinical atherosclerosis1,2,3,4 and cardiovascular disease (CVD) compared to the general population5,6. The increased cardiovascular risk in SLE cannot be fully explained by traditional cardiac risk factors alone7,8. Accumulating evidence indicates that inflammation plays a key role in development of atherosclerosis from plaque initiation to plaque rupture and thrombus formation9,10. SLE is a chronic inflammatory disease with increased levels of proatherogenic mediators11,12,13. Several studies suggest that disease-specific factors other than traditional risk factors may play an additional role in development of atherosclerosis and CVD in SLE8,14. Potent antiinflammatory medication provides survival benefits by reducing development of atherosclerosis and risk of coronary disease in patients with SLE14,15. Thus, it is likely that factors modulating the inflammatory response in SLE may affect development of atherosclerosis and CVD in SLE patients.
Mannose-binding lectin (MBL) is a liver-derived serum protein involved in innate immune defense16. It binds microorganisms and cellular debris through the carbohydrate recognition domain. Serum MBL can directly opsonize pathogens and enhance the activity of phagocytic cells or it can activate complement via the lectin pathway. Differences in MBL serum concentrations can partly be explained by structural variant alleles in the human MBL gene (MBL2) on chromosome 1017. The normal genotype is associated with the highest serum concentrations of MBL. Heterozygosity for MBL2 structural variant alleles causes an average 85%–90% drop in the serum concentration of functional MBL and homozygosity for structural variant alleles are devoid of functional MBL. The normal MBL2 allele is denoted A, and the common designation for the variant alleles is O. In addition to the effect of the structural allelic variants, polymorphisms in the promoter region of MBL2 gene affect serum levels of MBL. In particular, a base substitution at codon −221 (G to C; promoter allele X) is associated with lower serum concentrations of MBL.
Studies indicate an association between MBL deficiency and susceptibility for SLE18, and low MBL concentrations have been related to the clinical course of SLE, e.g., complicating infections19 and renal involvement20. In a followup study we observed that homozygosity for MBL2 variant alleles was associated with an increased risk of arterial thrombosis among patients with SLE21. This is in accord with another study where SLE patients with MBL deficiency had a 3.1 increased risk of CVD22. Studies of various populations have shown that the presence of dysfunctional alleles and low serum levels of MBL are associated with increased risk of atherosclerosis and coronary artery disease23,24,25,26.
Intima-media thickness of the common carotid artery (ccIMT) determined by high-resolution B-mode ultrasonography is a useful noninvasive anatomic measure of subclinical CVD and constitutes an excellent surrogate marker of generalized atherosclerosis and coronary artery disease27. Increased ccIMT has been observed in patients with SLE compared to controls1,4 and correlates with several traditional and nontraditional cardiovascular risk factors in SLE1,4,28,29.
Based on these findings we hypothesized that low-expressing MBL2 genotypes and low serum levels of MBL are associated with increased subclinical atherosclerosis measured by ccIMT in patients with SLE.
MATERIALS AND METHODS
Patients
Forty-one consecutive unrelated white Danish outpatients with SLE attending the Department of Rheumatology, Rigshospitalet, Copenhagen University Hospital, were included from August 2005 to July 2006. All patients fulfilled the American College of Rheumatology (ACR) classification criteria for SLE30. At inclusion, all patients were subjected to a detailed interview and underwent a clinical examination. Fasting serum samples were collected and stored at −80°C. Within 24 weeks of recruitment ccIMT was measured in all patients. The study was approved by the local scientific ethical committee (KF 01 261669). Written informed consent was obtained from each patient.
Clinical and laboratory assessment
Traditional cardiovascular risk modifiers included in this study were: sex, age, blood pressure calculated as mean blood pressure (1/3 × systolic blood pressure + 2/3 × diastolic blood pressure), smoking calculated as “pack-years” [mean number of packs (= 20 cigarettes) per day × number of years smoking], body mass index (BMI), total cholesterol, homeostatic model assessment of insulin resistance (HOMA1-IR) [(insulin (mU/l) × glucose (mmol/l))/22.5]31. Nontraditional cardiovascular risk modifiers were disease duration, highly sensitive C-reactive protein (hsCRP), SLE Disease Activity Index (SLEDAI)32, Systemic Lupus International Collaborating Clinics/ACR Damage Index (SDI)33, and treatment-ever with glucocorticoids or other immunosuppressants. All patients were interviewed and examined by the same physician. Clinical charts were used to verify information regarding treatment with antiinflammatory drugs.
Genotyping and detection of MBL serum concentration
Detection of MBL2 alleles was as described34. All patients were genotyped for the three MBL2 structural polymorphisms and for the promoter polymorphism in position —221 (X/X, X/Y, Y/Y). The following MBL2 extended genotypes were tested for: YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, and YO/YO. Serum concentrations of MBL were measured in a double ELISA based on a monoclonal anti-MBL antibody35. Low-expressing genotypes were defined to include XA/XA, YA/YO, XA/YO, and YO/YO based on the genotypes expressing the lowest MBL serum concentrations (Figure 1), while YA/YA and YA/XA were defined as high-expressing genotypes due to their associations with the highest MBL serum concentrations.
Serum concentrations of mannose-binding lectin (MBL) in relation to MBL2 extended genotypes in 41 patients with SLE. Data are presented as box plots: boxes represent 25th to 75th percentiles, lines within boxes the median, and the lines outside represent 10th and 90th percentiles.
*Outliers.
ccIMT measurement
Measurement of ccIMT was performed by using an Acuson Cypress ultrasound apparatus with a 7 MHz linear probe. A still picture of the common carotid artery proximal to the bulbus caroticus was produced, ensuring a distinct picture of intima and media on the far wall of the vessel (the intima-media). Thickness was measured 3 times using the electronic calipers of the ultrasound apparatus and the picture was digitally stored for future reference. Measurements were performed on both carotid arteries. A mean for both arteries was calculated and used in analysis. To ensure correct calibration of ccIMT measurements these were also performed in 10 healthy subjects (8 women, 2 men) with a median age of 44 years (range 25–72 yrs).
Statistical analysis
The differences in ccIMT between SLE patients and healthy individuals and between low-expressing and high-expressing MBL2 genotypes were examined by nonparametric Mann-Whitney test. Spearman’s rank correlation analysis was performed to examine the relation between ccIMT and MBL serum concentrations. Multivariable regression analyses were performed to examine the relationship among ccIMT, MBL2 low- and high-expressing genotypes, serum MBL, traditional and nontraditional risk factor variables. Due to the number of study subjects, the number of variables included into the regression model had to be limited, focusing on MBL, demographics, traditional cardiovascular risk factors, disease activity, and medication. To fit the linear regression model regarding linearity, variance homogeneity, and normal distribution, ccIMT was log-transformed. Processing of study data was done using SPSS 15. Statistical significance was defined as p value < 0.05.
RESULTS
Clinical and laboratory variables for all patients at inclusion are shown in Table 1.
Clinical and laboratory variables assessed in the cohort of 41 patients with systemic lupus erythematosus. Except where indicated otherwise, values are median [quartiles] (range).
The median ccIMT of SLE patients was 0.57 mm (range 0.37 to 0.98 mm), which was significantly thicker (p = 0.029) than in the healthy subjects, where median ccIMT was 0.46 mm (range 0.40 to 0.78 mm).
Median serum concentration of MBL was 0.85 mg/l (range 0 to 4.3 mg/l). Serum MBL varied with the extended MBL2 genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, and YO/YO) as shown in Figure 1. Seventeen patients had the low-expressing genotypes (XA/XA, YA/YO, XA/YO, and YO/YO) and 24 patients had the high-expressing genotypes (YA/YA, YA/XA). Variation in ccIMT according to low-expressing and high-expressing MBL2 genotype is shown in Figure 2. Using nonparametric Mann-Whitney test we found a significant difference in ccIMT between the low- and high-expressing MBL2 genotypes (p = 0.034). The difference in ccIMT remained significant in multivariable analysis adjusting for traditional and nontraditional cardiovascular risk modifiers (p = 0.049; Table 2).
Common carotid intima-media thickness (ccIMT) in relation to MBL2 low-expressing (17 patients) and high-expressing (24 patients) genotypes in 41 patients with SLE. Data are presented as box plots: boxes represent 25th to 75th percentiles, lines within boxes the median, and the lines outside represent 10th and 90th percentiles. Circles indicate outliers.
Results of multivariable regression analysis between common carotid intima-media thickness (ccIMT)*, MBL2 low-expressing genotypes, and traditional and nontraditional cardiovascular risk modifiers.
The relationship between ccIMT and serum concentrations of MBL is shown in Figure 3. ccIMT and serum MBL correlated negatively with a Spearman rho = −0.33 (p = 0.037). In a multivariate regression analysis we found that this negative association remained significant after adjustment for traditional and nontraditional cardiovascular risk modifiers (p = 0.042; Table 3).
Common carotid intima-media thickness (ccIMT) in relation to serum concentrations of mannose-binding lectin (MBL).
Results of multivariable regression analysis between common carotid intima-media thickness (ccIMT)*, serum MBL, and traditional and nontraditional cardiovascular risk modifiers.
DISCUSSION
Two studies have related MBL deficiency with CVD in SLE patients21,22. Since patients with SLE have increased incidence of atherosclerosis that cannot be explained solely by excess of traditional cardiovascular risk factors and since MBL deficiency has been associated with atherosclerosis and CVD in other populations23,24,25,26, we wanted to examine if MBL2 variant alleles and low serum MBL concentrations in SLE patients were associated with increased sub-clinical atherosclerosis measured by ultrasonography. Our results showed that ccIMT was increased in SLE patients compared to healthy individuals and that this increase was related to genetically determined low serum levels of MBL independently of the effects of traditional and nontraditional cardiovascular risk modifiers.
ccIMT ≥ 0.60 mm is a marker of generalized atherosclerosis36. In our study, the mean ccIMT was 0.62 mm, in accord with previous findings in SLE patients1,4. Several associations between increased ccIMT and traditional cardiovascular risk factors have been observed in patients with SLE1,4,28,29. In some studies disease duration29, SLEDAI37, and SDI38 have been associated with increased subclinical atherosclerosis in SLE patients, whereas other studies failed to find significant associations among disease duration, SLEDAI, SDI, and atherosclerosis in univariate and/or multivariate analysis28,39. Increase of ccIMT in SLE patients has also been shown to be associated with complement activation29 and proinflammatory high-density lipoprotein28. Moreover, treatment with antiinflammatory drugs (in particular prednisolone) was also associated with development of atherosclerosis in patients with SLE28,39. Due to the relatively low number of patients included in this study only a limited number of variables could be added into the multivariable analysis. Since a higher number of studies find significant associations among traditional cardiovascular risk factors, treatment with antiinflammatory drugs, and atherosclerosis in SLE patients, we chose to focus on these risk factor variables in our analysis. A priori we chose not to include SDI in the analysis since it is partly defined by atherosclerotic events, and therefore per definition could be expected to covary with ccIMT. Except for age, ccIMT was unrelated to other traditional and nontraditional cardiovascular risk factors in univariate (data not shown) and multivariate analyses in our study. It is likely that differences in study design, in risk factor variables, and in medication, age and duration of disease may influence the associations studied. The discrepancies in our results could also be due to uneven distributions of confounders not accounted for, which may include MBL.
MBL can enhance the clearance of pathogens, dying host cells, and cellular debris16, as well as circulating immune complexes40,41,42. Based on these capabilities several pathophysiological mechanisms may be suggested to explain the relation between deficiency of functional MBL and development of atherosclerosis in patients with SLE. A theory of infection in atherosclerosis has not been proven, but several studies have shown that chronic infections including periodontitis and infections with Helicobacter pylori and Chlamydia pneumoniae may be associated with development of atherosclerosis and cardiovascular disease43. As MBL deficiency is associated with increased risk of infections in SLE patients19; as infection with C. pneumoniae leads mainly to development and progression of severe coronary artery disease in patients with variation in the MBL gene44; and as homozygosity for MBL2 variant alleles is associated with greater impairment in flow mediated dilatation during infection in childhood24, it could be hypothesized that deficiency of MBL in SLE patients results in defective clearance of infectious material that aggravates ongoing inflammatory processes leading to development of atherosclerosis.
Endothelial apoptosis contributes to the loss of endothelial integrity and thereby to the initiation of atherosclerosis45. In patients with SLE the number of apoptotic circulating endothelial cells is increased, which correlates with the endothelial dysfunction observed in these patients46. Moreover, SLE patients produce a heterogeneous array of antibodies that bind to the endothelium47,48 and in vitro studies have shown that some of these antibodies induce endothelial apoptosis49. Lack of functional MBL may lead to decreased removal of apoptotic endothelial cells in patients with SLE. As high levels of circulating apoptotic endothelial cells may induce production of autoantibodies directed against endothelial cells, this could lead to dysfunction of the endothelium and enhance development of atherosclerosis in patients with SLE.
In contrast to our observations in SLE patients, the normal MBL2 genotype and high serum levels of MBL have also been shown to increase the risk of CVD in apparently healthy men50, in patients with rheumatoid arthritis (RA)51, and in patients with diabetes52; and high serum levels of MBL and the lectin pathway of complement initiates the inflammatory reaction seen in relation to ischemia reperfusion53,54. A differentiated role of MBL in atherosclerosis development was recently observed in patients with RA55. This paradox is probably a key issue in understanding the role of MBL in human disease. Thus, it is likely that a fine balance in the vessel wall during all stages of development of atherosclerosis determines whether MBL may be advantageous or disadvantageous. Under circumstances allowing the physiological opsonic activity of MBL, which is advantageous, lack of functional MBL may increase inflammation due to dysfunctional sequestration of harmful material from the vessel wall. On the other hand, if high levels of MBL mediate excessive complement activation the process may become detrimental. However, based on our results we are not able to determine causal relations. Experimental studies should be performed to examine the mechanisms by which MBL may influence the various stages of atherosclerosis in SLE patients and in other study populations.
Our results support the notion that genetically determined low levels of MBL are associated with the development of atherosclerotic disease in SLE. As our study population was relatively small, new studies based on larger study populations should be performed to confirm our data and to examine if our findings may be applicable in other disease settings and in healthy individuals.
Footnotes
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Supported by grants from The Danish Rheumatism Association, The Novo Nordisk Research Foundation, The Capital Region of Denmark, and Rigshospitalet, Copenhagen University Hospital.
- Accepted for publication April 26, 2010.
