Background and aims Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterised by thrombosis, obstetric complications and the presence of anti-phospholipid antibodies such as anti-β2GPI-Abs. These antibodies may set off the coagulation cascade via several mechanisms, including the induction of tissue factor (TF) expression. Vitamin D has recently emerged as an immunomodulator that might exert an anti-thrombotic effect. Therefore, we studied serum vitamin D levels in a cohort of APS patients, as well as the effect of vitamin D in an in vitro model of APS-mediated thrombosis.
Methods Serum vitamin D levels were measured in 179 European APS patients and 141 healthy controls using the LIAISON chemiluminescent immunoassay, and the levels were evaluated in conjunction with a wide spectrum of clinical manifestations. In an vitro model, anti-β2GPI antibodies were purified from four patients with APS to evaluate the expression of TF in activated starved human umbilical vein endothelial cells. The effect of vitamin D (1,25-dihydroxyvitamin D, 10 nm) on anti-β2GPI-Abs mediated TF expression was analysed by immunoblot.
Results Vitamin D deficiency (serum level ≤15 ng/ml) was documented in 49.5% of our APS patients versus 30% of controls (p<0.001) and was significantly correlated with thrombosis (58% vs 42%; p<0.05), neurological and ophthalmic manifestations, pulmonary hypertension, livedo reticularis and skin ulcerations. In vitro vitamin D inhibited the expression of TF induced by anti-β2GPI-antibodies.
Conclusions Vitamin D deficiency is common among APS patients and is associated with clinically defined thrombotic events. Vitamin D inhibits anti-β2GPI-mediated TF expression in vitro. Thus, vitamin D deficiency might be associated with decreased inhibition of TF expression and increased coagulation in APS. Evaluation of vitamin D status and vitamin D supplementation in APS patients should be considered.
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In recent decades, our knowledge of the metabolic functions of vitamin D has grown tremendously and its therapeutic roles have crossed many boundaries. Currently, vitamin D is no longer regarded only as a vitamin, essential for calcium and bone metabolism, but as a mediator of many non-calcaemic effects such as modulation of the immune response.1 Vitamin D is instrumental in combating certain microbial pathogens, as well as having a positive effect on cardiovascular diseases, malignancy, thrombosis and autoimmunity.2,–,5 Adequate levels of vitamin D can lead to a 7% reduction in all cause mortality, and relatively higher serum levels of 40 ng/ml are estimated to reduce the economic burden of overall morbidity by 10–16%.6 7 Vitamin D responses are mediated mainly through the vitamin D receptor (VDR), a nuclear receptor that binds to specific DNA sequence elements in vitamin D-responsive genes.8 VDRs are present in various organs and cell types such as monocytes, dendritic cells and lymphocytes. Thus, vitamin D might directly and indirectly affect the innate and adaptive arms of the immune system. Inhibition of maturation, proliferation and differentiation of these cells alongside the induction of immune tolerance by vitamin D has been documented.9 10
Furthermore, a close alliance between vitamin D and autoimmunity has been observed both in experimental models of autoimmune encephalomyelitis, rheumatoid arthritis, type 1 diabetes mellitus and in human autoimmune diseases.8 11 12 Specifically, lower serum levels of vitamin D or VDR gene polymorphisms are significantly more prevalent among patients with systemic lupus erythematosus (SLE),12,–,15 type 1 diabetes mellitus,16 rheumatoid arthritis,17 multiple sclerosis18 and autoimmune thyroid diseases.19 Moreover, we recently demonstrated an association between low levels of vitamin D and SLE disease activity,13 while others have reported a correlation with clinical manifestations such as fatigue.15
Antiphospholipid syndrome (APS) is a systemic autoimmune disease that may present either as a primary disease or secondary to other autoimmune diseases, predominantly SLE. It is characterised by the presence of anti-phospholipid antibodies, recurrent thrombosis, obstetric morbidity and various manifestations that may involve any organ or system.20 The anti-phospholipid antibodies, mainly those directed at β2GPI, are considered pathogenic and may cause the disease by interfering with homeostatic reactions that occur on the surface of vascular cells and platelets.20,–,22 Anti-β2GPI antibodies activate endothelial cells and monocytes, thereby inducing adhesion molecule and tissue factor (TF) expression.21 22 TF is a transmembrane protein of the class II cytokine and haematopoietic growth factor receptor family. The extracellular domain of TF serves as a receptor for activated factor VIIa, and interaction of TF with factor VIIa stimulates platelet activity and initiates blood coagulation.22 TF expression and upregulation in endothelial cells and monocytes is advocated as a pivotal step in anti-phospholipid antibody-mediated thrombosis.
Currently, data regarding serum vitamin D levels and the role of vitamin D in the pathogenesis of APS are lacking. Hence, in this study we measured serum vitamin D levels in patients with APS, both primary and secondary to SLE, and compared them with the levels in healthy controls. We evaluated the correlations between low levels of vitamin D and clinical manifestations of the disease. We further investigated the effect of vitamin D on anti-β2GPI-mediated TF expression.
Serum samples were collected from 179 patients of European origin who were diagnosed with APS according to the 2006 Sydney international consensus criteria,23 and 141 age- and sex-matched, healthy European controls. Our APS cohort was composed of 113 patients diagnosed with primary APS (pAPS) and 66 with APS secondary to SLE (sAPS).
Evaluation of clinical manifestations
Data on a wide spectrum of clinical manifestations were collected retrospectively, and then analysed and grouped according to major organs or systems involved as follows.
Thrombotic events included deep or superficial venous or arterial thrombosis, as well as evidence of pulmonary embolism. Pulmonary manifestations included pulmonary hypertension (defined by echocardiography or catheterisation), pleuritis, pulmonary haemorrhage, pneumonitis and pulmonary fibrosis. Skin manifestations included livedo reticularis and skin ulcerations. Neuro-psychiatric manifestations included epilepsy, migraine, cognitive impairment, multi-infarct dementia, encephalopathy, movement pathologies (ie, cerebellar ataxia), transverse myelopathy, Guillain–Barre syndrome, paresis, myasthenia gravis-like syndrome, neuropathy, depression and psychosis. Ophthalmic manifestations included sicca syndrome, chorioretinitis, microaneurysms, glaucoma, cataracts and blepharitis. Articular manifestations included arthralgia and arthritis. Obstetric manifestations included fetal loss, intrauterine growth retardation and pre-eclampsia/eclampsia. Haematological manifestations included thrombocytopenia, anaemia/haemolytic anaemia, disseminated intravascular coagulation and leucopenia. Renal manifestations included renal failure (defined by elevated creatinine levels, proteinuria above 0.5 g/24 h or evidence of glomerulopathy. Cardiac manifestations included valve thickening and dysfunction, unstable angina, pericarditis, myocarditis and myocardial infarction.
Vitamin D measurement
We used the LIAISON vitamin D Assay (310600; DiaSorin, Saluggia, Italy) commercial kit to measure serum concentration of vitamin D in of patients and controls.24 Briefly, this method for quantitative determination of 25-hydroxyvitamin D is a direct, competitive chemiluminescent immunoassay. A specific antibody to vitamin D is used to coat magnetic particles (solid phase) and vitamin D is linked to an isoluminol derivative. During incubation, 25-hydroxyvitamin D is dissociated from its binding protein, and competes with labelled vitamin D for binding sites on the antibody. After incubation, the unbound material is removed with a wash cycle. Subsequently, the starter reagents are added and a flash chemiluminescent reaction is initiated. The light signal is measured by a photomultiplier as relative light units and is inversely proportional to the concentration of 25-hydroxyvitamin D present in calibrators, controls or samples. Previously, it has been hypothesised that vitamin D status can be divided into ranges of serum 25-hydroxyvitamin D concentrations termed deficient, insufficient or sufficient. Traditionally, a serum 25-hydroxyvitamin D value of less than 10 ng/ml or less than 20 ng/ml has been considered to be the cut-off point for deficiency. A recent study defined serum 25-hydroxyvitamin D values <15 ng/ml as deficient, 15–30 ng/ml as insufficient and >30 ng/ml as sufficient.25,–,27 In the current study deficiency of vitamin D was defined accordingly as serum levels below 15 ng/ml.
In vitro studies: evaluation of the effect of vitamin D on anti-β2GPI antibody-mediated TF expression
Anti-β2GPI antibodies were purified from four APS patients that presented with a thrombotic event and high titres of anti-cardiolipin and anti-β2GPI antibodies, utilising a β2GPI affinity purified column as previously described.28 Anti-β2GPI were affinity purified on a β2GPI column. β2GPI was affinity purified from healthy blood donor plasma using a heparin column (Pharmacia HealthCare, GE HealthCare Bio Sciences AB, Uppsala, Sweden) followed by a nickel column (Pharmacia HealthCare) and protein-G column (Sigma-Aldrich, St Louis, MO, USA). The purity of β2GPI was confirmed by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot. A β2GPI column was constructed employing an NHS-HiTrap column (Pharmacia HealthCare) according to the manufacturer's protocol. Sera from APS patients were loaded (separately) on the β2GPI column. The anti-β2GPI Abs were eluted with 2.5 M glycine-HCl pH 2.5, neutralised by Tris pH 9 and dialysed against culture medium. Commercial IgG (Jackson, West Grove, Pennsylvania, USA) was used as IgG control. Human umbilical vein endothelial cells (HUVECs) primary cultures were purchased from Lonza (Walkersville, Maryland, USA). The cells were maintained in commercial endothelial cells basal medium-2 (EBM-2) (Lonza), supplemented with vascular endothelial growth factor. The cells were used during passages 1–4 until 80% confluent. Before the experiments, the cells were starved for 8 h in EBM-2 with 0.5% fetal calf serum and no additional growth factors. Following this starvation period, the cells were incubated with anti-β2GPI antibodies (10 µg/ml) for an additional 7–8 h (activation stage). Thereafter, the cells were lysed with lysis buffer (20 mM Tris–HCl (pH 7.5), 1% SDS, protease inhibitor cocktail, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF) (all the reagents were from Sigma-Aldrich). For Western blot analyses, 25 µg of the lysates were loaded on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, Massachusetts, USA) followed by blocking with 10% non-fat milk overnight at 4°C. TF expression in the lysate was analysed by immunoblot, utilising mouse anti-human CD142 (TF) IgG1 (9010-5059; AbD Serotec, Oxford, UK). Following striping, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected by mouse monoclonal IgG1 Abs. Vitamin D (1,25-dihydroxyvitamin D; Sigma-Aldrich) at different concentrations was added to HUVECs during the activation stage (with anti-β2GPI antibodies). This was followed by analysis of the expression of TF as specified above. All doses of 1,25-dihydroxyvitamin D decreased the expression of TF while the most inhibitory concentration was found to be 10 nm (figure 1). Others have also reported 10 nm of 1,25-dihydroxyvitamin D to be highly potent in upregulating VDR in vitro,29 as well as affecting the phenotype of monoblastic cells.30 1,25-Dihydroxyvitamin D was dissolved in ethanol; therefore control cultures were also set up in ethanol 0.1%.
A comparison of prevalence rates between the groups was performed by χ2 and Fisher exact tests (two-tailed), as appropriate. Continuous variables are expressed as mean±SD, and were compared between groups by Student t test (two-tailed). For all tests, p value <0.05 was considered statistically significant.
Vitamin D levels in APS patients versus controls
Mean serum levels of vitamin D were significantly lower among APS patients compared with controls: 16.7±9 versus 21.6±10 ng/ml, respectively (p<0.001). Vitamin D deficiency (serum vitamin D <15 ng/ml) was documented in 87/176 (49.5%) of the APS patients versus 42/141 (30%) of controls (p<0.001), whereas higher levels of vitamin D were more prevalent in healthy subjects (figure 2). The mean levels of vitamin D differed between patients with primary and secondary disease and were 18±9 ng/ml among pAPS versus 14±8 ng/ml in sAPS (p=0.004). However, upon individual analysis, both levels were significantly lower than that for the controls (p=0.002 for pAPS and p<0.001 for sAPS). Age at the time of the study, as well as age at diagnosis of disease, correlated with low serum vitamin D levels only among sAPS patients. The current cohort of APS patients comprised 80% women and 20% men (table 1). In this study sex was not associated with vitamin D levels: the mean levels of vitamin D among women was 16.6±9 versus 17.5±9.6 among men (p=0.6). In the entire cohort 51% of women and 53% of men were diagnosed with vitamin D deficiency; in the group of patients with sAPS vitamin D deficiency was documented in 58% of women and 50% of men (p>0.6 for both).
The prevalence of APS manifestations arrayed according to vitamin D levels
Data on clinical manifestations of APS were gathered and grouped by organ involvement to better reflect clinical disease. Lower serum levels of vitamin D (<15 ng/ml) correlated with thrombotic, pulmonary, ophthalmic, skin and neurological events. Other manifestations, such as obstetric, joint, haematological, renal and cardiac events (table 2), were not correlated with levels of vitamin D, and neither did the presence of anti-phospholipid antibodies.
The correlation between APS manifestations and low serum vitamin D levels did not differ between patients with pAPS and sAPS. Thrombotic events were documented in 77% of pAPS patients with vitamin D levels below 15 ng/ml, compared with 52.5% in those with higher levels (p=0.01). Similarly, venous thrombosis was documented in 60% versus 41% (p=0.05), superficial thrombophlebitis in 28% versus 12% (p=0.05), pulmonary emboli in 24% versus 8% (p=0.04) and pulmonary hypertension in 10% versus 0% (p=0.03) of pAPS patients with serum vitamin D levels below and above 15 ng/ml, respectively. Multi-infarct dementia was documented in 8% of pAPS patients with serum vitamin D levels below 15 ng/ml compared with none of those presenting with higher levels (p=0.04). In contrast, ophthalmic manifestations were more common among sAPS patients with vitamin D deficiency (42.5% versus 10% (p=0.01)).
Vitamin D suppressed anti-β2GPI-mediated TF expression
In our in vitro model, the anti-β2GPI antibodies were purified from APS patients that presented with a thromboembolic event. These antibodies were added to HUVECs and induced the expression of TF, in contrast to added human IgG (figure 3A). The addition of 10 nm of vitamin D, diluted in ethanol, suppressed the expression of TF in each of the four patients evaluated (figure 3A for patients 1 and 2 and figure 3B for patients 3 and 4). This was not observed following the addition of ethanol. The expression of the control protein, GAPDH, was not affected by vitamin D (figure 3).
In the current study, we found vitamin D deficiency to be significantly more prevalent among APS patients compared with healthy controls. This is in accordance with our former observation of low vitamin D levels among 160 patients with APS.12 In addition, we found vitamin D levels to be significantly lower among patients with APS secondary to SLE, compared with those diagnosed with pAPS. Recently, we reported in a large cohort of patients with SLE that low serum concentrations of vitamin D were inversely correlated with disease activity.13 An association was also demonstrated between fatigue and vitamin D concentrations in SLE patients.31 In the current cohort of sAPS patients, vitamin D deficiency was documented in 57% of subjects and correlated with older age but not with sex (ie, 58% of women and 50% of men with sAPS presented with serum vitamin D below 15 ng/ml) (table 1). Similarly to our previous study no linkage was detected between vitamin D levels and sex among SLE patients.13
To the best of our knowledge, this is the first report of a significant association between vitamin D deficiency and clinical manifestations of APS. Of note vitamin D insufficiency (serum level <30 ng/ml) has been commonly documented in healthy subjects worldwide, as well as in the current study (figure 1). Therefore, we analysed the association between very low levels (<15 ng/ml), which defines vitamin D deficiency, and APS manifestations. An inverse correlation between vitamin D levels and thrombosis was observed in our cohort. Interestingly, most other manifestations that were found to be related to vitamin D deficiency in this study (such as pulmonary hypertension, livedo reticularis, skin ulceration and certain neurological events) may also be attributed to the existing coagulopathy of APS.32 This link between vitamin D and thrombosis was previously reported in other conditions. A recent Cox regression analysis of more than 29 000 subjects in southern Sweden found that the risk of venous and arterial thrombosis increased by 50% during the winter compared with other seasons.33 Furthermore, women who were more exposed to the sun had a significantly lower risk of thrombosis.33 Another study reported fewer thrombotic events among cancer patients treated with high-dose calcitriol. The effect of calcitriol in this study was independent of previous thrombotic events and the use of anti-thrombotic agents.34 Further support for this notion was indirectly observed in patients treated with hydroxychloroquine (HCQ). This medication in high doses has an anti-thrombotic effect in SLE and APS patients that may be attributed to the inhibition of platelet-activation signal transduction or to decreased antibody-mediated thrombosis.35 Surprisingly, higher levels of vitamin D were documented in patients treated with HCQ.36 Thus, one may speculate that ultraviolet light exposure or the administration of supplements that improve vitamin D status may in turn generate anticoagulation properties.
Activation of endothelial cells and monocytes by anti-phospholipid antibodies results in over-expression of TF and adhesion molecules, which are considered to be major mechanisms leading to thrombosis in APS.35 In our study we found that vitamin D is a potent inhibitor of anti-β2GPI antibody-mediated TF expression in endothelial cells. A similar effect of vitamin D was documented by others utilising tumour necrosis factor or lipopolysacharide-induced TF expression on monocytes.37 38 Anti-β2GPI antibodies affect endothelial cells via activation of nuclear factor κB and a signalling cascade that ultimately induce pro-coagulation. Recently, this activation was found to be dependent on toll-like receptor (TLR)-4 signalling, and a decreased incidence of anti-β2GPI mediated thrombosis was documented in TLR-4-deficient mice.39 40
Vitamin D may decrease the proliferation and activation of endothelial cells and monocytes,37 41 as well as the expression of TF in monocytes37 38 and endothelial cells. Furthermore, it has been documented that vitamin D is able to suppress expression of TLRs such as TLR-4. Reduced TLRs expression is accompanied by impaired nuclear factor κB translocation to the nucleus and by reduced TLR-dependent signal transduction.42
Taken together, the data support a plausible role for vitamin D in ameliorating mechanisms linked to thrombosis in APS.
Our study has several limitations, including the retrospective collection of clinical data, and the lack of data on nutrition, previous vitamin supplementation and seasonality differences. Moreover, variability of vitamin D levels may be documented even in the same subject, and could not be assessed in this study. However, in recent years, treatments with anticoagulation and anti-platelet aggregation of APS-associated thromboembolic events were linked with serious adverse events, reduced quality of life and recurrences in spite of treatment. Thus, there is an urgent need for novel, safer and more efficient modalities to be used either alone or in combination with current therapy.35 Our data suggest an inverse association between vitamin D and thrombosis among APS patients, alluding to a possible beneficial effect of vitamin D supplementation.
Large, randomised, controlled studies that address the long-term effects of vitamin D supplementation as well as the mechanisms by which vitamin D inhibits the expression of TF at the levels of transcription and translations are clearly needed.
In conclusion, we found serum vitamin D levels to be significantly lower among APS patients, and associated with certain manifestation of this syndrome (ie, thrombosis). In addition, in a complementary in vitro study, the addition of vitamin D exerted an anti-thrombotic effect via inhibition of endothelial cells expression of TF. Thus, we can also speculate that in vivo vitamin D deficiency results in reduced inhibition of TF expression, thereby promoting coagulation. A potential beneficial effect of vitamin D supplementation in APS is thus suggested.
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Sheba Medical Center, Israel, and fulfilled the ethical guidelines of the Declaration of Helsinki (Edinburgh, 2000).
Provenance and peer review Not commissioned; externally peer reviewed.
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