Low bone density and low serum levels of soluble RANK ligand are associated with severe arterial calcification in patients with Takayasu arteritis

M. C. Bezerra, G. D. Calomeni, V. F. Caparbo, E. S. Gebrim1, M. S. Rocha1 and R. M. R. Pereira

Divisions of Rheumatology and 1 Radiology, School of Medicine, University of São Paulo, São Paulo, Brazil.

Correspondence to: R. M. Rodrigues Pereira, Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo, 455 – 3 andar – Reumatologia, Sala 3107, São Paulo, SP, 01246–000 Brazil. E-mail: rosamariarp{at}yahoo.com


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. Takayasu arteritis (TA) is a chronic inflammatory disorder affecting the aorta and its branches. Vascular calcification has been described in 29–54% of cases of TA, although its aetiology remains unknown. Recently the osteoprotegerin/RANKL/RANK system has emerged as an important contributing factor to atherogenesis and osteogenesis. Our aim is to investigate the association between vascular calcification, bone mineral density (BMD) and the osteoprotegerin/RANK/RANKL system in TA.

Methods. Thirty pre-menopausal female TA patients and 30 age- and sex-matched controls were studied. BMD was measured by dual X-ray absorptiometry. Arterial calcification in TA patients was analysed by computed tomography in thoracic and abdominal sites. Serum levels of osteoprotegerin and soluble receptor activator of nuclear factor {kappa}B ligand (sRANKL) were quantified by enzyme-linked immunosorbent assay.

Results. Patients with severe arterial calcification showed lower BMD values than controls in lumbar spine (0.965 ± 0.055 vs 1.126 ± 0.153 g/cm2, P = 0.009) and total body (0.993 ± 0.065 vs 1.085 ± 0.082 g/cm2, P = 0.019). In contrast, TA patients without calcification presented BMD values similar to controls (P>0.05). Interestingly, lower serum levels of sRANKL (1.89 ± 2.35 vs 2.80 ± 2.23 pg/ml, P = 0.031) and a longer disease duration (12.20 ± 6.61 vs 3.56 ± 5.33 yr, P = 0.004) were observed in TA patients with severe calcification compared with patients without calcification.

Conclusions. Severe arterial calcification in TA is associated with low values of BMD and sRANKL, reinforcing the possible link between bone and vascular disease.

KEY WORDS: Takayasu arteritis, Bone mineral density, sRANKL, Arterial calcification


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Takayasu arteritis (TA) is a chronic inflammatory disease that affects large-calibre arteries. Vasculitis is probably triggered by an autoimmune process in which the inflammatory cell infiltrate is characterized by activated T cells, macrophages, multinucleated giant cells and cytokines [1].

Some studies have demonstrated calcification of the aorta wall in 29–54% of TA cases, and it has been associated with a poor prognosis [2]. Atherosclerosis and osteoporosis are commonly associated, especially in older women with oestrogen deficiency and in autoimmune diseases, and a close relation exists between the clinical course of arterial calcification and osteoporosis [3–5]. Various hypotheses have been implicated: oestrogen deficiency, vitamin D metabolism, dyslipidaemia and the osteoprotegerin (OPG)/receptor activator of nuclear factor {kappa}B ligand (RANKL)/RANK systems [5, 6].

RANKL is highly expressed by T cells and osteoblast/stromal cells, and also exists in active soluble forms, either secreted from T cells or proteolytically cleaved from cell surfaces. Cell response to RANKL depends on the level of expression of its receptor RANK, but also on the presence of its decoy receptor, OPG [7]. Immune mechanisms involving activated T cells (the source of RANKL) and antigen-presenting dendritic cells (the target for RANKL) are implicated in vascular inflammatory diseases, and probably in TA. It has been speculated that RANKL and its antagonist, OPG, represent an important cytokine system in vascular biology [8]. As TA is a chronic inflammatory disease that primarily affects the aorta, we believe that it will be interesting to study atherosclerosis related to bone metabolism.

The objective of the present study was to evaluate the relationship between vascular calcification and osteogenic mechanisms by analysing bone mineral density (BMD) and the OPG/RANKL/RANK system in TA.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The study was conducted on 30 consecutive pre-menopausal women with TA according to American College of Rheumatology (ACR) criteria, who were followed up at the vasculitis out-patient clinic. Thirty age-matched healthy females served as controls. The local ethics committees approved the study and written informed consent was obtained from patients and controls. The following data were collected from all patients: identification, clinical manifestation, disease duration, risk factors for coronary disease, use of glucocorticoids, clinical and laboratory disease activity. Clinical activity was defined based on the presence of new onset or worsening of fever or musculoskeletal problems; vascular ischaemia or inflammation such as claudication, diminished or absent pulse, bruit, carotidynia or asymmetric blood pressure. High erythrocyte sedimentation rate (ESR) and/or C-reactive protein (CRP) levels in the absence of infection characterized laboratory activity. Laboratory parameters were considered to be altered when total cholesterol >5.18 mmol/l, low-density lipoprotein (LDL) cholesterol ≥3.367 mmol/l, high-density lipoprotein (HDL) cholesterol <0.9065 mmol/l, triglycerides >2.26 mmol/l, CRP >5 µg/dl and ESR >13 mm.

Bone densitometry
Bone mass was determined in patients and controls by dual X-ray absorptiometry in the lumbar spine (L1–L4), femoral neck and whole body using a Hologic QDR 2000 densitometer.

Chest and abdominal computed tomography
The 30 TA patients underwent chest and abdominal computed tomography (CT). The exams were performed with a helicoidal high speed tomograph (General Electric Medical System, Milwaukee, OR, USA) using a routine protocol for abdominal CT (120 kV, 200 mA; thickness 10 mm, spacing 10 mm). The aim of the exam was to determine the presence of arterial calcifications in the region of the ascending and descending aorta, suprarenal and infrarenal abdominal aorta, coeliac trunk, superior and inferior mesenteric artery, and iliac and renal arteries. Calcification was quantified as severe when the entire vessel diameter was affected, mild when the vessel diameter was partially involved and absent when there was no radiological evidence of calcification.

Laboratory examinations
Serum levels of calcium, cholesterol and triglycerides, ESR and high-sensitivity CRP were measured using routine laboratory methods. Serum for the determination of OPG, soluble RANKL (sRANKL) and 25-hydroxyvitamin D was collected from patients and controls after fasting and stored at –80°C for later measurement.

25-Hydroxyvitamin D was measured by chemiluminescence using reagents from Nichols Institute Diagnostics (San Clemente, CA, USA).

Serum OPG was assayed by ELISA (Biomedica, Vienna, Austria), which uses a monoclonal anti-OPG antibody to capture OPG from serum. Serum levels of uncomplexed (free) sRANKL were measured by an enzyme immunoassay (Biomedica), according to the manufacturer's instructions [9].

Statistical analysis
Data are reported as mean ± S.D. and per cent. Anthropometric variables and BMD values were compared between patients and controls by the Student's t-test. In patients with TA, the following quantitative variables were submitted to multiple linear regression analysis using L1–L4 and whole body BMD as dependent variables: disease duration, risk factor for coronary disease, cholesterol, triglycerides, ESR, CRP, calcium, vitamin D, OPG and sRANKL. T scores <–1 S.D. (osteopenia/osteoporosis) and ≥–1 S.D. (normal) were correlated with the categorical variables clinical disease activity, laboratory activity, use of simvastatin and past and present use of glucocorticoids by applying Fisher's exact test.

Patients with severe calcification and without calcification were compared regarding cholesterol, calcium and vitamin D using a parametric test; and regarding disease duration, coronary disease risk factors, triglycerides, ESR, CRP, OPG and sRANKL using the Mann–Whitney U-test. The variables glucocorticoid use and clinical and laboratory disease activity were analysed by Fisher's exact test. P values <0.05 were considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
No difference in anthropometric data was observed between patients and controls (P>0.05). In TA, the mean disease duration was 6.96 ± 7.24 yr, 80% had taken glucocorticoids, 57% used this drug at the time of the study and 13% were taking simvastatin (the only statin available for our patients). There was a higher frequency of laboratory disease activity (80%) compared with clinical activity (30%). Sixty-seven per cent of these patients presented at least one risk factor for coronary disease. Total and LDL cholesterol were elevated in 53.3 and 47% of TA patients, respectively.

Lumbar spine (0.967 ± 0.130 vs 1.058 ± 0.137 g/cm2, P = 0.011) and whole body (0.990 ± 0.081 vs 1.037 ± 0.091 g/cm2, P = 0.041) BMD was significantly lower in patients than in controls. No differences in OPG, sRANKL or 25-hydroxyvitamin D were observed between patients and controls.

Multiple linear regression analysis using L1–L4 and whole body BMD as dependent variables and disease duration, coronary disease risk factors and serum cholesterol, triglycerides, calcium, ESR, CRP, 25-hydroxyvitamin D, sRANKL and OPG as independent variables showed no statistical significance for TA.

Patients with osteopenia/osteoporosis (T score <–1 S.D.) of L1–L4 and whole body were compared with normal patients (T score ≥–1 S.D.) in terms of clinical and laboratory activity, simvastatin use, and present and past use of glucocorticoids. A significant difference was only observed for past use of glucocorticoids in patients with osteoporosis/osteopenia of the whole body (P = 0.02).

Arterial calcification was present in 50% of the patients, and most frequently affected the descending aorta (33.3%). Calcifications were also observed in the suprarenal (26.7%) and infrarenal (23.3%) abdominal aorta, ascending aorta (16.7%), right (6.7%) and left (3.3%) common iliac artery, and coeliac trunk (3.3%).

Analysis of patients with severe arterial calcification and comparison with age-matched controls showed significantly lower L1–L4 and whole BMD. No significant difference in BMD in these regions was observed between patients without arterial calcification and controls (Table 1).


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TABLE 1. Bone mineral density (BMD) in Takayasu arteritis (TA) patients without and with severe calcification and in age-matched controls

 
Comparison of patients with severe calcification and those without calcification in terms of disease duration, coronary disease risk factors, clinical and laboratory activity, corticosteroid use, cholesterol, triglycerides, calcium, vitamin D, ESR, CRP, OPG and sRANKL revealed a significant difference for disease duration (P = 0.004) and sRANKL (P = 0.031) (Table 2).


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TABLE 2. Clinical and laboratory parameters of patients with Takayasu arteritis (TA) without and with severe arterial calcification

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The present study demonstrated a reduction of BMD in patients with TA, especially those with severe arterial calcification, suggesting an association between bone and vascular disease.

Schulz et al. [10], studying 2348 post-menopausal women, showed that aortic calcifications were inversely related to BMD and directly related to fractures, suggesting that aortic calcifications are a strong predictor for osteoporosis and fragility fractures. Similarly, Pennisi et al. [9] demonstrated low bone density and abnormal bone turnover in 36 patients with serious carotid and femoral atherosclerotic involvement. Vascular calcification and osteoporosis frequently occur together and share many of the same risk factors, indicating a pathophysiological link between the two diseases [3, 11, 12].

In the present study, 80% of TA patients showed inflammatory activity, suggesting persistent inflammatory vascular disease even in the presence of therapy. Several cytokines with regulatory effects on immune function appear to contribute to bone homeostasis by enhancing bone resorption. Also, some of these [interleukin-1 (IL-1), tumour necrosis factor-{alpha} (TNF-{alpha})] are implicated in atherosclerotic calcification regulated by OPG/RANK/RANKL [13, 14].

In our study, others factors associated with vascular calcification such as oestrogen deficiency, excess vitamin D, glucocorticoid use and dyslipidaemia showed no significant difference between patients with and without calcifications.

Controversy surrounds oestrogen and its effects on cardiovascular disease. Recently, the Women's Health Initiative randomized controlled trial showed that hormone replacement therapy increases the risk for cardiovascular disease [15]. We selected only young pre-menopausal patients in order to exclude possible interference of oestrogen deficiency or replacement as another factor to atherosclerosis and osteogenic process.

There is evidence that excess vitamin D induces both atherosclerosis and osteoporosis in humans and in laboratory animals [16]. No difference in 25-hydroxyvitamin D levels was observed between TA patients and healthy controls or between patients with and without calcifications.

Glucocorticoids have been associated with an increased risk of low BMD, fractures and atherosclerosis [5]. In our study, a higher frequency of osteoporosis/osteopenia associated with the use of glucocorticoids was only observed for the whole body region, but there was no significant difference in the use of this drug between patients with severe arterial calcification and those without calcification. Interestingly, glucocorticoid is known to decrease the production of OPG and the expression of RANKL in the skeletal and vascular systems, but in our study there was no difference between patients and controls in serum levels of these proteins. This finding suggests that the steroid effect was probably counterbalanced by a more relevant inflammatory stimulus (IL-1, TNF) which may have induced an increase in OPG production by endothelial and osteoblast/stromal cells [13, 14].

An altered lipid metabolism is associated with both bone remodelling and the atherosclerotic process [17]. Although 47% of our TA patients showed high LDL cholesterol levels, no difference in these values was observed between TA patients without and with severe calcification.

Similarly, there was no difference in OPG levels in patients with or without calcification and controls. The oestrogen-replete status of all TA patients and controls may have been a relevant contributing variable influencing the OPG level [7, 14].

Alternatively, this absence of association with most laboratorial measurements may be a consequence of the study design which evaluates only a picture of a recent disease status.

In contrast, arterial calcification and BMD are more a reflection of long-term processes that may have been operational in the past. In fact, a longer disease duration was found to be significant for the occurrence of calcification. The persistent immune-inflammatory activity observed in these patients, with the presence of activated immune cells (a source of RANKL) and a lot of cytokines that increase the expression of RANKL which could interact with its receptor RANK in endothelial cells and osteoclasts, certainly played the most important role in the atherosclerotic and osteogenic process.

In the present study, significantly lower levels of sRANKL were observed in patients with severe calcification compared with those without calcification. Similarly, Schoppet et al. [18] found that low serum sRANKL levels were associated with the presence of coronary artery disease. RANKL has been demonstrated in the extracellular matrix surrounding the calcium mineral deposit of plaques [19]. Moreover, RANKL transcripts were detected in the calcified arteries of OPG-deficient mice. These findings suggest that RANKL may be involved in the regulation of mineralization in atherosclerotic lesions. On the other hand, RANKL is a known cytokine involved in the formation and activation of osteoclasts and consequent promotion of bone resorption [7, 14, 20].

Taking into consideration that only free sRANKL was measured in the present study, our findings suggest that either the majority of RANKL is interacting with RANK in osteoclast and endothelial cells or else there is an insufficient counter-regulatory mechanism. In both conditions, the interruption of RANKL binding seems to be a potential therapeutic target to prevent bone resorption and atherosclerotic process in TA.


    Acknowledgments
 
This study was supported by FAPESP (#03/08916–2). G.D.C. was the recipient of a FAPESP fellowship (#04/01009–2).

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 14 June 2005; Accepted 12 July 2005





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