Cardiovascular risk in systemic lupus erythematosus—evidence of increased oxidative stress and dyslipidaemia

S. L. Nuttall1,, S. Heaton2, M. K. Piper2, U. Martin1 and C. Gordon2

Departments of
1 Clinical Pharmacology and
2 Rheumatology, University of Birmingham, Birmingham, UK


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. Systemic lupus erythematosus (SLE) is associated with severe and premature cardiovascular disease, which is not explained by traditional risk factors alone. This study aimed to investigate markers of oxidative stress, lipid metabolism and inflammation as potential cardiovascular risk factors in women with SLE.

Methods. Venous blood samples were taken from 53 female Caucasian patients with SLE and from healthy age- and sex-matched controls. Samples were analysed for markers of oxidative stress, lipid metabolism [including low-density lipoprotein (LDL) subfraction profile] and C-reactive protein (CRP).

Results. Female SLE patients had an atherogenic lipid profile characterized by raised total cholesterol and triglycerides, and the presence of small, dense LDL subfractions compared with healthy controls. These changes were associated with increased oxidative damage and a moderately raised CRP.

Conclusions. The results provide evidence for free radical and inflammatory activity in SLE and suggest potential targets to reduce the risk of cardiovascular disease in these patients.

KEY WORDS: Lupus, Oxidative stress, LDL subfractions, Cardiovascular risk.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an autoimmune, multisystem disorder, usually presenting in pre-menopausal women, which appears to be increasing in incidence [1]. Although the acute mortality has declined, many SLE patients die prematurely and the leading cause of death in patients with a disease duration of more than 5 yr is coronary artery disease (CAD) [2]. In the Framingham Offspring Study, women with SLE aged 35–44 yr were at 52 times greater risk of myocardial infarction than healthy women of a similar age [3]. Traditional risk factors only partly explain the high mortality rates and therefore other factors have to be considered, particularly the contribution of SLE itself. These include an elevated homocysteine level [4], or the combination of oxidative stress, inflammation and the presence of antiphospholipid antibodies [2], which promote atherosclerosis by increasing the uptake of oxidized low-density lipoproteins (LDL).

LDL oxidation is a key factor in the initiation of atherosclerosis [5], and hence the development of CAD. Small, dense LDL appear to be particularly pro-atherogenic; they are easily oxidized, are retained in the arterial intima and are readily taken up by resident macrophages creating foam cells, an early development in the formation of atherosclerotic lesions. Women with SLE have an atherogenic lipid profile with raised cholesterol and triglycerides [2], a combination commonly associated with an increase in smaller, denser LDL subfractions. These subfractions, when subjected to oxidative stress, which would be expected in an inflammatory disorder like SLE, could be a key risk factor and account, at least in part, for the high incidence of cardiovascular disease.

We describe a cross-sectional study assessing the LDL subfraction profile (assessed by calculating an ‘LDL score’) and markers of oxidative stress (total antioxidant capacity and lipid hydroperoxides) in women with SLE compared with healthy female controls. We also investigated and correlated changes in traditional lipid abnormalities (i.e. total cholesterol and triglycerides) and C-reactive protein (CRP) concentrations, which are undetectable in most healthy subjects.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Subjects
An unselected group of 53 Caucasian female patients with a diagnosis of SLE (median age 40 yr, range 20–64 yr) were recruited from the lupus clinic at the Queen Elizabeth Hospital, Birmingham. Our population was limited to Caucasian females as they were our largest population and our ethnic minorities were too few to analyse [6]. All patients fulfilled the American Rheumatism Association (ARA) classification criteria for SLE [7]. The mean duration of disease was 10.6 yr (range 0–25 yr) and none had renal failure (mean±S.D. serum creatinine 84.8±12 µmol/l). Eight of the patients were positive for antiphospholipid antibodies. Patients were taking prednisolone (43 patients, median dose 7.8 mg, range 2.5–30 mg), other immunosuppressants (hydroxychloroquine, azathioprine and cyclophosphamide) (n=45), statins (3), ACE inhibitors (7), ß-blockers (2) and aspirin (16). Comorbidities included angina and/or myocardial infarction (n=3), cardiomyopathy (2), cerebrovascular disease (3), diabetes (1) and proteinuria (5) as defined by the SLICC/ACR damage index [8].

Disease activity was assessed by the British Isles Lupus Assessment Group (BILAG) Index, which is a computerized index for measuring disease activity in SLE routinely used in our lupus clinic [9]. Each of eight organ-based systems is given an alphabetic score from A–E. A and B indicate active disease whereas C represents stable mild disease. D represents a system previously affected but currently inactive, while E indicates the relevant system was never involved in the disease process. For the purposes of this study we noted whether or not the patient had ever scored a BILAG A or B in the systems that were considered most relevant to the risk of atherosclerosis, namely the heart, lungs, arteries and kidneys, or had scored A (most active disease) in any other system.

Fifty-three healthy Caucasian women (median 38 yr, range 21–64) were recruited as a control group. They had no major medical or surgical disease, and were on no medication. They were matched for age and sex and none had clinical evidence of cardiovascular disease or diabetes.

The study was approved by the South Birmingham Local Research Ethics Committee and all participants gave written informed consent according to the Declaration of Helsinki.

Analysis
Venous blood from non-fasting patients and controls was analysed for markers of lipid metabolism and oxidative stress. Serum total cholesterol and triglycerides were determined by standard colour spectrophotometry and CRP by radioimmunoassay. The LDL subfraction profile was determined by disc polyacrylamide gel electrophoresis [10]. Briefly, this method separates the LDL into seven different bands (denoted 0–6) according to their mobility along the gel. An LDL score was then calculated by assessing the area under the curve for each of the bands (the higher the score, the greater the proportion of smaller, denser LDL subfractions, normal range 1.5) [10].

Serum total antioxidant capacity was analysed by enhanced chemiluminescence [11]. This method depends on the continuous production of a free radical-mediated luminescent light signal, which is temporarily stopped when a sample containing antioxidants is added. The time taken for the signal to return is directly proportional to the antioxidant content. Plasma lipid hydroperoxides (a marker of oxidative damage) were determined spectrophotometrically by the ferrous-oxidation of xylenol orange [12]. The specific marker of lupus activity, C3d, was measured by enzyme-linked immunosorbent assay [13].

Statistics
Statistical differences between groups were determined by Student's unpaired t-test. Pearson linear regression was used to assess correlation between variables and logistic regression for assessing the effects of treatment. Significance was taken to be P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The lipid metabolism results for SLE patients and healthy controls are presented in Fig. 1Go. Serum total cholesterol and triglyceride concentrations were significantly increased in women with SLE compared with healthy counterparts. Furthermore, the LDL score was markedly elevated, indicating a shift towards smaller, denser, more atherogenic LDL particles [10].



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FIG. 1. Markers of lipid metabolism and oxidative damage (LHP) in patients with SLE and healthy controls. Values expressed as mean±S.D. LHP, p < 0.0001; LDL, p < 0.0001; Cholesterol, p < 0.01; Triglycerides, p < 0.001.

 
The changes in lipid metabolism in patients with SLE were associated with an increased concentration of lipid hydroperoxides (the marker of oxidative damage) and a non-significant trend towards lower total antioxidant capacity (TAC) [mean (95%CI): 354.2 (331.7–376.7) vs 381.7 (358.1–405.3) µmol/l trolox eq., P=0.1].

There were no significant differences in any markers between SLE patients with and without comorbidities except for lipid hydroperoxides, which were elevated in those patients with comorbidities (Table 1Go). The significant changes in lipid metabolism and oxidative stress observed between patients and healthy controls persisted even when patients with comorbidities were excluded (Table 1Go).


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TABLE 1. Markers of lipid metabolism and oxidative stress in SLE patients with and without comorbidities compared with healthy controls

 
Total cholesterol was positively correlated with lipid hydroperoxides, LDL score, triglycerides and the dose of prednisolone. The LDL score was also correlated with triglyceride concentrations, lipid hydroperoxides and with CRP [6.93 (4.26–9.60) mg/l, upper limit of normal 5 mg/l], a general marker for inflammatory activity. Thus the more active the inflammation (even low grade, as measured by CRP), the more atherogenic the lipid profile. A more specific marker of lupus activity, C3d, was positively correlated with triglycerides and total cholesterol. There were no further correlations, however, with any other specific markers of lupus activity (C3/C4 or anti-ds DNA antibodies). The correlations between these markers are presented in Table 2Go.


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TABLE 2. Associations between markers of lipid metabolism, oxidative stress, inflammation and lupus activity

 
The use of immunosuppressants and aspirin were positively correlated with TAC (r=0.30 and 0.31, respectively, P < 0.01) suggesting an anti-inflammatory and/or antioxidant potential for these therapies. None of the markers was correlated with concurrent disease activity, as assessed by the BILAG index [9], or antiphospholipid status.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The incidence of SLE appears to be increasing [1] and the main cause of death in later disease is CAD [3]. The pathogenesis of CAD in this population is likely to be multifactorial. Previous studies have confirmed the involvement of traditional cardiovascular risk factors (such as lipid levels, smoking and the presence of diabetes) and have identified more novel risk factors (for example, homocysteine and antiphospholipid antibodies) [2, 4]. The present study has provided evidence that the increased risk may be attributed to three additional important risk factors.

First, the atherogenic lipid profile demonstrated in patients with SLE can be further characterized; the elevated plasma cholesterol and triglyceride concentrations are associated with a higher LDL score not previously identified in these patients. This high LDL score indicates the presence of smaller, denser LDL particles compared with controls, which accelerate atherosclerosis. Similar observations can be found in rheumatoid arthritis patients with high cardiovascular-related mortality [14]. Second, these LDL particles are more likely to be oxidized, and therefore taken up by macrophages, because the patients are in a state of greater oxidative stress, with reduced antioxidant capacity and raised plasma lipid hydroperoxides. Third, inflammatory disorders have been increasingly implicated in the development of CAD [1518]. SLE is a chronic inflammatory disease in which inflammatory and immune disease processes are likely to contribute to the accelerated atherosclerosis observed in this disease [19].

We have demonstrated a significant correlation between inflammatory activity, as determined by the CRP, LDL score and concentration of lipid hydroperoxides. The association was apparent even though the biochemical markers (CRP) suggested that the inflammatory activity was low grade. This is of particular interest as previous studies have demonstrated a link between small increases in CRP and carotid artery disease in these patients [20]. Svenungsson et al. [21], in a separate study, also identified the involvement of more traditional risk factors for the presence of cardiovascular disease in SLE, which were also associated with carotid intima-media thickness, used as a surrogate marker of the degree of atherosclerosis. In contrast to our findings they reported no difference in lipid metabolism between patients without cardiovascular complications and controls. Our study demonstrated abnormalities prior to the onset of complications in SLE, and those patients with comorbidities had a further increase in oxidative damage. However, care must be taken in interpreting the latter observation as the number of patients with cardiovascular disease was small.

The results of the present study are compatible with the results of other published studies showing enhanced oxidative stress in rheumatic diseases [22] and evidence of antibodies to oxidized LDL in SLE patients [23], although the predictive capacity of the antibodies alone must be interpreted with caution as the results can differ substantially between laboratories and there is a large inter-subject variation. In a trial by Iuliano et al. [24], the presence of antiphospholipid antibodies in patients with SLE was associated with an increase in lipid oxidation, as measured by isoprostane formation from the free radical-mediated oxidation of arachidonic acid. The authors suggest that lipid peroxidation may underlie the antiphospholipid syndrome, although a cause–effect relationship was not confirmed by their results. Some studies have provided evidence that certain drug therapies, such as the long-term use of corticosteroids, accelerate the progression of atherosclerosis [25]. Prednisolone may be pro-atherogenic as a result of its effects on lipids. It increases total cholesterol and very low- (VLDL), low- (LDL) and high-density lipoprotein (HDL)-cholesterol, especially in patients taking more than 30 mg of prednisolone daily, but it may also be anti-atherogenic, particularly at lower doses [21, 26].

Hydroxychloroquine has been shown to reduce total cholesterol, LDL-cholesterol and triglycerides in several studies and has beneficial lipid-lowering properties in patients taking regular corticosteroids [2729]. However, whether hydroxychloroquine can prevent cardiovascular events has yet to be demonstrated. The role of cytotoxic agents in promoting or preventing cardiovascular complications has not been studied. Azathioprine can be hepatotoxic and may induce fatty liver with overflow secretion of VLDL and in susceptible patients can induce a secondary mixed hyperlipidaemia. Cyclosporin A can increase LDL cholesterol levels and theoretically may promote atheroma [30]. However, the steroid-sparing effects of these agents may reduce the insulin resistance, hypertriglyceridaemia and the increase in total cholesterol, VLDL- and LDL-cholesterol associated with high-dose steroid therapy, while also preventing long-term renal damage. This is important as renal impairment and persistent proteinuria (especially in the nephrotic range) promotes hypercholesterolaemia.

We did not find an association with our markers of dyslipidaemia and the use of non-steroid immunosuppressive drugs in this study, in which only five patients had chronic nephrotic-range proteinuria and none had renal impairment. Even more importantly, we did find that immunosuppressive therapy was associated with reduced oxidative stress as measured by total antioxidant capacity. Our results were not dependent on disease duration, in agreement with previous work [21]. Data are needed from clinical trials currently in progress to assess the relative effects of azathioprine, cyclosporin A and steroid dose on lipids in SLE patients as most current data are from renal transplant recipients [30] and the number of patients on each drug in the present study was too small to assess independently.

Statins inhibit the 3-hydroxy-3-methylglutaryl coenzyme A reductase. As a result they can reduce the serum level of LDL-cholesterol. Although they are extensively prescribed to prevent cardiovascular mortality and morbidity in the non-lupus population, there have been no published trials involving lupus patients treated with statins to date. At present there is increasing interest in testing statins in SLE patients as they may have a disease-modifying role through their effects on inflammation in addition to their effects on lipids [31]. Only three of the patients in this study were taking statins for hypercholesterolaemia, so it was not possible to analyse the specific effects of statins on lipids or oxidative stress in this analysis.

SLE is a chronic disease interspersed with episodes of acute disease flares. These episodes are likely to result in damage to the arterial endothelium causing endothelial dysfunction, as we have previously demonstrated [32], a known predictor of future cardiovascular events [33]. Therefore, it is important to be able to highlight modifiable risk factors early in the development of the disease. The present study has identified some of these risk factors in a cohort of female patients with SLE. They have an atherogenic lipid profile with a preponderance of small, dense LDL subfractions, which are associated with free radical-mediated oxidative damage, and evidence for the importance of inflammatory processes that may be involved in the development of CAD. The results of this study may partly explain the increased risk of premature CAD in SLE, and therefore suggest some potential therapeutic interventions, such as with non-steroid immunosuppressive and anti-inflammatory agents, and with antioxidants, which may reduce the cardiovascular morbidity and mortality associated with this disease. Such approaches may be particularly relevant to patients requiring steroid therapy who are unable to tolerate statins or hydroxychloroquine. Further clinical trials comparing such therapeutic strategies are required urgently.


    Acknowledgments
 
We are grateful to M. J. Kendall (Department of Clinical Pharmacology) and P. A. Bacon (Department of Rheumatology) for their support and critical review of the manuscript, to J. Skan for database support and to A. Garfield for measurement of C3d. This study was funded in part by grants from LUPUS UK and the Arthritis Research Campaign. There are no competing interests.


    Notes
 
Correspondence to: S. L. Nuttall, Clinical Investigation Unit, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK. E-mail: s.nuttall{at}bham.ac.uk Back


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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 8 April 2002; Accepted 28 November 2002