Rheumatology

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Rheumatology 2001; 40: 401-405
© 2001 British Society for Rheumatology

Antibodies against oxidized low-density lipoproteins in systemic sclerosis

A. L. Herrick, K. J. Illingworth, S. Hollis¹, J. M. Gomez-Zumaquero² and F. J. Tinahones²

University of Manchester Rheumatic Diseases Centre, Hope Hospital, Salford,
¹ Medical Statistics Unit, Lancaster University, Lancaster, UK and
² Hospital Regional Carlos Haya, Malaga, Spain

	Abstract

Top Abstract Introduction Patients and methods Results Discussion References

 
Objective. To investigate whether circulating concentrations of antibodies against oxidized low-density lipoproteins (LDL) are increased in patients with systemic sclerosis (SSc).

Methods. Oxidation of LDL and anti-oxidized LDL antibodies were measured in 26 patients with limited cutaneous SSc (LCSSc), in eight patients with diffuse cutaneous SSc (DCSSc) and in 24 healthy control subjects. Results were adjusted for age, sex and smoking.

Results. Binding to oxidized LDL was increased in patients with both limited and diffuse cutaneous disease (geometric mean 0.35 and 0.39 optical density units respectively) compared with controls (0.28) (P=0.03 and P=0.01 respectively). Circulating concentrations of anti-oxidized LDL were increased only in patients with diffuse SSc (geometric mean 0.22 optical density units) compared with controls (geometric mean 0.16, P=0.02).

Conclusion. These preliminary findings lend further weight to the concept that oxidation of LDL contributes to the vascular pathology of SSc, particularly in patients with diffuse cutaneous disease. Prospective longitudinal studies are required to investigate whether anti-oxidized LDL antibodies may be a marker of vascular damage in SSc.

KEY WORDS: Lipoproteins, Anti-oxidized LDL antibodies, Oxidant stress, Systemic sclerosis.

	Introduction

Top Abstract Introduction Patients and methods Results Discussion References

 
Vascular abnormalities are a prominent feature of systemic sclerosis (SSc), and it has been suggested that SSc is primarily a vascular disease [1]. In recent years there has been increasing interest in the concept of oxidant stress being one of the factors responsible for the vascular injury [2]; we and other investigators, using a number of different techniques, have reported increased free radical activity in patients with SSc [3–9].

Of particular interest, Bruckdorfer et al. [7] reported that low-density lipoproteins (LDL) isolated from patients with SSc demonstrated increased susceptibility to oxidation. Oxidation of LDL is thought to be a key factor in the promotion of atherogenesis [10]. This raises the question of whether patients with SSc are at increased risk of LDL oxidation and whether this is a major factor in the development of their vascular disease. This is especially relevant now that patients with SSc are believed to be at risk of large vessel as well as small vessel disease [11].

Oxidized LDL is more immunogenic than native LDL and it has been suggested that antibodies directed against oxidized LDL predict the progression of carotid atherosclerosis [12]. Increased circulating concentrations of anti-oxidized LDL have been reported in patients with systemic lupus erythematosus and with antiphospholipid syndrome [13–15] as well as in diabetes [16]. Very recently, Simonini et al. [17] reported that concentrations of anti-oxidized LDL were increased in patients with SSc. The aim of the present pilot study was to investigate further whether patients with SSc had increased circulating concentrations of anti-oxidized LDL compared with control subjects and whether these concentrations differed between patients with limited cutaneous and diffuse cutaneous disease. If anti-oxidized LDL concentrations are elevated in patients with SSc, then future studies could address whether these might have predictive value for vascular disease severity.

	Patients and methods

Top Abstract Introduction Patients and methods Results Discussion References

 
Patients
Twenty-four healthy control subjects (two male, 22 female; median age 50 yr, range 29–69 yr), 26 patients with limited cutaneous SSc (LCSSc) (three male, 23 female; median age 53 yr, range 26–79 yr) and eight patients with diffuse cutaneous SSc (DCSSc) (four male, four female; median age 53 yr, range 42–73 yr) were studied. The median skin score (modified Rodnan technique; 17 sites, maximum score 3 for each site, as previously described [18]) in the patients with limited cutaneous disease was 5 (range 0–19) and in the patients with diffuse cutaneous disease it was 18 (range 3–37). Three of the controls, four of the patients with limited cutaneous disease (one of whom smoked cigars) and one of the patients with diffuse cutaneous disease were smokers. All patients with SSc were attending the rheumatology clinic at Hope Hospital, Salford, and full clinical details, including autoantibody status, were available for all patients (Table 1).

View this table: [in this window] [in a new window]   TABLE 1. Clinical features of all patients and those with and without raised concentrations of anti-oxidized LDL antibodies

 

Methods
Blood was collected using minimal stasis and atraumatic venepuncture into 10-ml glass bottles. The blood was stored at room temperature for at least 1 h before centrifugation at 2000 g, 4°C for 10 min. Approximately 3 ml serum was removed and stored in 7-ml bijou tubes at -70°C. Samples were later shipped on dry ice to Spain for processing.

LDL isolation
LDL was isolated from pooled plasma of healthy fasting human donors by density-gradient ultracentrifugation. The LDL was then dialysed against phosphate-buffered saline (PBS; 0.14 M NaCl/0.01 M phosphate buffer) at 4°C for 30 h.

Oxidization of LDL
Malondialdehyde (MDA)-modified LDL (MDA-LDL) was used as an antigen for detecting antibodies to oxidized LDL. Oxidized LDL was prepared by incubating the LDL for 3 h at 37°C with 0.5 M MDA at a constant ratio of 100 µl/mg of LDL. The 0.5 M MDA was generated from MDA bis-dimethylacetal by acid hydrolysis. The reaction was stopped by adjusting the pH to 7.4 with 1 M NaOH. After conjugation, MDA-LDL was extensively dialysed against PBS.

Anti-oxidized LDL antibodies
Microtitre plates for the determination of anti-oxidized LDL antibodies (using an oxidized LDL-specific ELISA) were coated with either native LDL or with MDA-LDL, both at 10 µg/ml in PBS. The plates were incubated for 2 h at 37°C and overnight at 4°C. After washing four times with PBS, plates were blocked with 1% bovine serum albumin (BSA)/PBS for 2 h at room temperature. Serum samples were diluted 1:100 in 1% BSA/PBS and incubated for 3 h at room temperature. After washing, alkaline phosphatase-conjugated anti-human IgG (Sigma Immuno Chemicals, Poole, UK), diluted 1:1000 in 1% BSA/PBS, was added. It was then left for 3 h at room temperature. p-Nitrophenyl-phosphate (Sigma), 1 mg/ml in 500 mM carbonate buffer containing 1 mM MgCl₂ (pH 9.8), was used as substrate. The absorbance was read at 405 nm. Results were expressed as optical density. Binding of antibodies to oxidized LDL was calculated by subtracting the binding of native LDL from binding to MDA-LDL.

Lag phase
The susceptibility of LDL to in vitro oxidation was assessed essentially by the technique described by Esterbauer et al. [19]. Isolated LDL was kept in darkness under nitrogen for 4 days at 4°C before determination of susceptibility to oxidation. The LDL preparation was then dialysed against a 100-fold volume of 0.02 mol/l phosphate buffer (pH 7.4)/0.16 mol/l NaCl (dialysis buffer) purged with nitrogen for 15 h at 4°C in darkness. The buffer was changed three times. EDTA (ethylenediamine tetraacetic acid)-free LDL was diluted in dialysis buffer to a final concentration of 25 µg/ml and oxidation was initiated by addition of a freshly prepared aqueous solution of copper sulphate to a final concentration of 1.66 µmol/l. The kinetics of LDL oxidation were monitored by changes in 234-nm absorbance at 30°C on a spectrophotometer equipped with a six-position automatic sample changer. The dienes formed during LDL oxidation produce an absorption spectrum with a distinct peak at 234 nm with essentially no interindividual variation; the initial absorbance at 234 nm was taken as the baseline and the change in absorbance was recorded every 5 min for 4 h. The absorbance curve at 234 nm was divided into three phases: a lag phase, a propagation phase and a decomposition phase. The lag phase was defined as the tangent of the slope of the absorbance curve in the propagation phase with the baseline, and was expressed in minutes [20].

Statistical analysis
Anti-oxidized LDL antibody measurements and lag time were log-transformed to achieve normality, and are presented as the geometric mean and coefficient of variation. Analysis of covariance was used to compare each group with the control simultaneously, with adjustment for age, sex and smoking.

	Results

Top Abstract Introduction Patients and methods Results Discussion References

 
These are summarized in Table 2 and Fig. 1.

View this table: [in this window] [in a new window]   TABLE 2. Binding to native LDL and to oxidized LDL (MDA-LDL), anti-oxidized LDL and lag time in patients with SSc and control subjects

 

View larger version (11K): [in this window] [in a new window]   FIG. 1. Anti-oxidized LDL antibodies (optical density units) in healthy control subjects, patients with DCSSc and patients with LCSSc.

 

Binding to native LDL
Although a small number of patients with each subtype of disease demonstrated increased binding to native LDL, there were no significant differences between subject groups.

Binding to MDA-LDL
This was raised in both SSc subgroups compared with healthy control subjects (P=0.03 for LCSSc and P=0.01 for DCSSc).

Anti-oxidized LDL
Compared with healthy control subjects, anti-oxidized LDL antibodies were significantly elevated in patients with DCSSc (P=0.02) but not in patients with LCSSc (P=0.21) (Fig. 1).

Lag times
These were similar in all groups.

Clinical features of patients with concentrations of anti-oxidized LDL antibodies higher than the control range
Clinical features of the nine patients with concentrations of anti-oxidized LDL antibodies above the highest control value (0.321 optical density units) are compared with those of the other 25 patients in Table 1. There were no obvious clinical differences between groups, other than there being only one male patient with a high value. The results in Table 2 are adjusted for sex as well as for age and smoking.

	Discussion

Top Abstract Introduction Patients and methods Results Discussion References

 
Our results suggest that circulating concentrations of antibodies to oxidized LDL are raised in a proportion of patients with SSc; differences from healthy controls reached statistical significance for the subgroup of patients with diffuse cutaneous disease. Binding to oxidized LDL was significantly increased in both SSc subgroups. These findings therefore support the hypothesis that LDL oxidation may be increased in patients with SSc. However, this phenomenon is not universal, at least not at all stages in the disease process, because the majority of patients with SSc demonstrated levels well within the reference range.

Our results therefore differ from those of Simonini et al. [17], who found that levels of anti-oxidized LDL antibodies were highest in patients with limited cutaneous disease and in patients with early disease. However, a significant point is that both studies support the concept that oxidation of LDL contributes to the vascular pathology of SSc. There were some methodological differences between the study of Simonini et al. and our own: Simonini et al. used Ca²⁺-oxidized LDL and in our study we used oxidized MDA-LDL.

We and others [6, 21] have reported reduced circulating levels of antioxidants in patients with SSc, and these may predispose patients towards oxidant stress and thus LDL oxidation. Perhaps surprising was that levels of anti-oxidized LDL antibodies were higher in patients with DCSSc than in those with LCSSc, because patients with LCSSc are generally considered to have more severe vascular disease, often in association with anticentromere antibodies [22, 23]. This more severe vascular disease in patients with the limited cutaneous subtype has recently been demonstrated by laser Doppler imaging [24]. On the other hand, patients with DCSSc tend to have more internal organ involvement and so the higher concentrations in this group might reflect their more generalized disease. The increased concentrations of anti-oxidized LDL antibodies provide a further rationale for the use of antioxidants in patients with SSc [25, 26].

We did not find that the oxidative capacity of LDL was increased in patients with SSc and therefore our findings contrast with those of Bruckdorfer et al. [7], who found this increase in both limited cutaneous and diffuse cutaneous disease. It may be relevant that in the study by Bruckdorfer et al., 20 of 47 patients (43%) had diffuse cutaneous disease as opposed to only eight of 34 (24%) in the present study. Oxidation lag times were found by Bruckdorfer et al. to be more reduced in patients with diffuse disease, and in our study there was a trend for the patients with diffuse disease to have shorter lag times.

Although this was a small pilot study allowing only limited conclusions, its findings lend further support to the hypothesis that oxidative stress with oxidative modification of LDL contributes to the pathogenesis of SSc. Key issues arising from our study are: (i) whether the measurement of anti-oxidized LDL antibodies can be used as a marker of vascular injury and therefore of disease activity or severity; and (ii) whether levels of anti-oxidized LDL antibodies predict the severity of microvascular or macrovascular involvement of disease.

These questions can only be addressed in prospective longitudinal studies, which should include patients with primary Raynaud's phenomenon. Markers of vascular involvement in SSc are badly needed; none of those currently used are ideal [27], and this has hampered clinical trials to identify treatments that favourably influence the progression of SSc-related vascular disease [26]. If high levels of anti-oxidized LDL antibodies are shown to be predictive of vascular disease severity, then perhaps measures to reduce vascular risk could be intensified in that cohort of patients found to be at particular risk.

	Notes

 
Correspondence to: A. L. Herrick, University of Manchester Rheumatic Diseases Centre, Hope Hospital, Salford M6 8HD, UK.

	References

Top Abstract Introduction Patients and methods Results Discussion References

 

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Submitted 29 February 2000; revised version accepted 25 October 2000.
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