Clinical Rheumatology https://doi.org/10.1007/s10067-018-4322-9
ORIGINAL ARTICLE
Dickkopf-1 (Dkk-1) serum levels in systemic sclerosis and rheumatoid arthritis patients: correlation with the Trabecular Bone Score (TBS) Barbara Ruaro 1 & Andrea Casabella 1,2 & Sabrina Paolino 1 & Carmen Pizzorni 1 & Massimo Ghio 1 & Chiara Seriolo 2 & Luigi Molfetta 2 & Patrizio Odetti 2 & Vanessa Smith 3,4 & Maurizio Cutolo 1 Received: 22 August 2018 / Revised: 25 September 2018 / Accepted: 1 October 2018 # International League of Associations for Rheumatology (ILAR) 2018
Abstract The aim of this research was to determine any correlations between Dickkopf-1 serum levels (Dkk-1, a natural inhibitor of the Wnt signaling pathway promoting osteoclastogenesis) and the Trabecular Bone Score (TBS), in systemic sclerosis (SSc) and rheumatoid arthritis (RA) patients. It also aimed at determining any difference in Dkk-1 serum levels between SSc and RA patients and a control group (CNT) of healthy subjects. A prospective study was carried out in 60 SSc and 60 RA patients and 60 CNT. Dkk-1 serum levels were evaluated by the ELISA method (Quantikine Human Dkk-1 Immunoassay, R&D System, Minneapolis, USA). The severity of microvascular damage was evaluated by nailfold videocapillaroscopy (NVC patterns: BEarly,^ BActive,^ BLate^), in the SSc patients. TBS analysis and bone mineral density (BMD, g/cm2) were measured in all patients/subjects. The SSc patients showed higher Dkk-1 serum levels than RA (p < 0.004) and CNT (p < 0.0001) patients. SSc patients, showing the BLate^ NVC pattern had statistically higher Dkk-1 serum levels than patients with either the BActive^ or BEarly^ (p < 0.004) patterns. Only in the BLate^ NVC pattern group of SSc patients was there a significant negative correlation between Dkk-1 and TBS values (p < 0.0001). The increased Dkk-1 serum levels and decreased TBS values observed suggest a diffuse bone damage in SSc patients with advanced disease, as demonstrated by the concomitant presence of the BLate^ NVC pattern. Moreover, the bone remodeling in SSc seems even higher than that in RA patients. Keywords Dickkopf-1 . Osteoporosis . Rheumatoid arthritis . Systemic sclerosis . Trabecular Bone Score
Introduction Dickkopf-1 (Dkk-1), a bone mass remodeling regulator, is a soluble protein secreted in the bone microenvironment and detectable in the circulation. Dkk-1 is a natural inhibitor of Barbara Ruaro and Andrea Casabella contributed equally to this work. * Barbara Ruaro
[email protected] 1
Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine (Di.M.I.), Polyclinic San Martino Hospital, University of Genova, Viale Benedetto XV, no. 6, 16132 Genoa, Italy
2
Centro di Ricerca su Osteoporosi e Patologie Osteoarticolari CROPO, Di.M.I., University of Genova, Genoa, Italy
3
Department of Rheumatology, Ghent University Hospital, Ghent, Belgium
4
Department of Internal Medicine, Ghent University, Ghent, Belgium
the Wnt signaling pathway that has been hypothesized to be involved in promoting osteoclastogenesis through the suppression of osteoprotegerin [1–5]. The Wnt/β-catenin pathway signaling has been identified as a key promoter of osteoblastogenesis, leading to new bone formation [1–6]. Numerous studies have evaluated Dkk-1 levels in rheumatic diseases such as rheumatoid arthritis (RA) and ankylosing spondylitis (AS), and significantly higher levels were observed in these patients than those in normal controls, suggesting that Dkk-1 might well play a role in the pathogenesis of these diseases [7–10]. Indeed, Dkk-1 seems involved in systemic sclerosis (SSc), both in joint remodeling/ectopic ossification and fibrosis [11–14]. A negative correlation between a reduced bone microarchitecture, evaluated by the Trabecular Bone Score (TBS), and the progression of the microvascular damage tested by nailfold videocapillaroscopy (NVC) has been demonstrated [15]. This result confirmed that microvascular damage is correlated with SSc clinical complications, including bone impairment [15–18].
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The objectives of our study were as follows: (i) to determine if there are any correlations between Dkk-1 serum levels and various NVC patterns (i.e., BEarly,^ BActive,^ and BLate^) in SSc patients; (ii) to compare Dkk-1 serum concentrations in SSc and RA patients and healthy subjects (CNT); and (iii) to evaluate any correlations between Dkk-1 serum levels and bone quality (TBS evaluation) in SSc patients. To the best of our knowledge, this is the first study to evaluate the Dkk-1 serum levels in SSc patients with different NVC patterns, aimed at establishing if there is a correlation between Dkk-1 serum levels and TBS in SSc and RA patients and CNT.
groups. Dkk-1 serum levels were measured by enzyme-linked immunosorbent assay (ELISA) (Quantikine Human Dkk-1 Immunoassay, R&D System, Minneapolis, USA).
The Trabecular Bone Score The TBS is an index of bone quality derived from dual-energy. Dual-energy X-ray absorptiometry (DXA) analysis was performed, as previously reported [15]. The lumbar spine L1-L4 TBS (unit-less) was calculated on each spine DXA examination by TBS iNsight (Medimaps group/GE Healthcare Needham, MA, USA, software version 2.1.0.0). A normal range for TBS values in postmenopausal women has been proposed: a TBS of ≥ 1.350 is considered normal; a TBS between 1.200 and 1.350 is considered consistent with partially degraded microarchitecture and a TBS of ≤ 1.200 defines degraded microarchitecture [15, 22–24].
Methods Study population A total of 180 women (60 SSc patients, 60 RA patients, and 60 healthy controls) in the postmenopausal period were recruited during routine clinical practice in our Rheumatology Department, from November, 2012 to November, 2015. All three groups had routine osteoporosis screening, according to our routine clinical practice. A complete medical history was collected and a clinical examination performed for all study participants (see Table 1). Particular attention was paid to any history of vertebral fractures. The SSc patients met either LeRoy’s criteria for the classification of early SSc or the ACR/EULAR 2013 criteria for SSc [19, 20]; the RA patients fulfilled the ACR/EULAR 2010 criteria for RA [21]. A total of 85% of the RA patients were under prednisone treatment at a dose of 2.5 to 5 mg/day. Our prospective study was approved by the local Ethical Committee. The inclusion criteria were patients with a diagnosis of SSc or RA. The exclusion criteria were being on a drug regimen that could potentially influence bone turnover, such as bisphosphonate, no stable drug regimen for at least 2 months prior to study entry, and a glucocorticoid dosage of > 5 mg prednisone/day, being affected by secondary causes of osteoporosis.
Bone parameters The overall metabolic activity of the bones was assessed by blood tests. The parathyroid hormone (PTH), 25 hydroxyvitamin D (25(OH)D), serum alkaline phosphatase (ALP), calcium, and phosphorus were evaluated for all three
Bone mineral density Bone mineral density (BMD, g/cm2) of the lumbar spine (L1L4) and left hip (femoral neck; Ward’s triangle; trochanter; total hip) was analyzed using DXA scan (Lunar Prodigy, GE Lunar, Madison, WI, USA) in all 180 subjects, as previously reported [15]. The DXA test uses two X-ray beams with different energy levels, which are aimed at the patient’s bones. Once the soft tissue absorption has been subtracted out, the BMD can be determined by the bone absorption of each beam. The weight, height, age, and gender of each patient were used to estimate the BMD (expressed as g/cm2 and standard deviation scores) and compared to the control group’s BMD values. BMD values were expressed as a T-score; this number shows the amount of bone present compared to a young adult of the same gender with peak bone mass [21–23]. A score above − 1 is considered normal in BMD T-scores obtained at the femur and lumbar spine, a score between − 1 and − 2.5 is classified as osteopenia (low bone mass), and a score below − 2.5 is defined as osteoporosis [25–27].
Nailfold videocapillaroscopy NVC was performed in the SSc patients to determine the correct microangiopathy pattern (i.e., BEarly,^ BActive,^ or BLate^), using an optical probe fitted with a × 200 contact lens, connected to an image analysis software (Videocap, DS Medica, Milan, Italy) [16–18, 28, 29]. NVC was performed only in SSc patients, as no specific microvascular lesions have been described in RA [16–18, 28–30]. The results showed that the patterns had an equal distribution, i.e., 20 SSc patients had an BEarly^ pattern; 20, an BActive^ pattern; and another 20, a BLate^ NVC pattern.
Clin Rheumatol Table 1 Characteristics of the systemic sclerosis (SSc) and rheumatoid arthritis (RA) patients and control group (CNT)
SSc, n = 60
RA, n = 60
Control, n = 60
p value
Age, years (SD)
56.9 (7.3)
57.6 (7.1)
56.6 (8.2)
> 0.05
BMI, kg/m2 (SD)
21.9 (1.8)
22.7 (2.9)
22.9 (3.1)
> 0.05
Age of menopause, years (SD) Disease duration, years (SD)
51.8 (2.3) 8.6 (6.1)
51.3 (1.7) 14.6 (6.3)
52.2 (2.8) NA
> 0.05 NA
Raynaud duration, years (SD)
10.5 (3.2)
NA
NA
NA
25(OH)D, ng/ml (SD) Dkk-1, pg/ml (SD)
12.7 (3.2) 3038 (771.3)
14.2 (7.6) 2737 (594.2)
13.9 (5.4) 2044 (521.7)
< 0.001 < 0.001
TBS, unit-less (SD)
1.102 (0.019) 1.018 (0.291)
0.998 (0.042) 0.934 (0.162)
1.461 (0.162) 1.268 (0.587)
< 0.001 < 0.001
− 1.615 (1.300) 0.742 (0.102)
− 1.140 (0.187) 0.668 (0.141)
0.138 (1.158) 0.923 (0.229)
< 0.001 < 0.001
− 1.920 (0.908) 0.582 (0.124)
− 1.540 (1.031) 0.403 (0.253)
− 0.554 (1.067) 0.677 (0.345)
< 0.001 < 0.001
− 2.634 (0.902) 0.686 (0.273) − 1.417 (0.976) 0.845 (0.294) − 1639 (0,952)
− 2.310 (1.087) 0.578 (0.209) − 1.237 (2.577) 0.753 (0.288) − 1.232 (1.203)
− 1.178 (1.139) 0.712 (0.152) 0.094 (1.022) 1.123 (0.209) − 0.097 (0.983)
< 0.001 < 0.001 < 0.001 < 0.001 < 0.001
Lumbar spine (L1-L4) BMD, g/cm2 (SD) T-score (L1-L4) Femoral neck BMD, g/cm2 (SD) T-score (femoral neck) Ward’s triangle BMD, g/cm2 (SD) T-score (Ward’s triangle) Trochanter BMD, g/cm2 (SD) T-score (trochanter) Total hip BMD, g/cm2 (SD) T-score (total hip)
ALP bone, bone alkaline phosphatase; NA, not applicable
Statistical analysis Statistical analyses were carried out by the GraphPad PRISM, version 5.02. Nonparametric tests were performed for the statistical analysis. The Mann-Whitney U test was used to compare unpaired groups of variables and the Kruskal-Wallis test was used to compare continuous variables to nominal variables with more than two levels. The Spearman rank correlation test was used to search for any relationships between variables, along with linear regression tests. Any p values below 0.05 were considered statistically significant. The results are reported as averages, along with standard deviation (SD).
Results Dkk-1 levels were statistically higher in SSc patients with the BLate^ NVC pattern than in those with the BActive^ and BEarly^ (p < 0.0001), and higher serum levels were observed in SSc patients than in RA patients (p < 0.0001) or CNT (p < 0.004) (Table 2). Only in the BLate^ NVC pattern group of SSc patients was there a significant negative correlation between Dkk-1 and TBS values (p < 0.001); no correlations were observed in patients with either the BActive^ or BEarly^ patterns or in RA and CNT groups. There were no significant correlations between Dkk-1 and DXA and T-score, in any skeletal area (respectively p > 0.05), in any study group.
A negative correlation between Dkk-1 levels and the duration of Raynaud’s phenomenon (years expressed) (p < 0.01) was observed, while there was no correlation between Dkk-1 and disease duration in SSc or RA patients. SSc patients had a statistically significant correlation between Dkk-1 and 25 hydroxyvitamin D (p = 0.01), no significant correlation with PTH, bone alkaline phosphatase, calcium, or phosphorus (p > 0.05). Lumbar spine TBS was significantly lower in SSc patients than that in the CNT (p < 0.001). No significant differences in TBS values were observed between the SSc and RA (p > 0.05) groups. TBS values were statistically higher in those SSc patients that had an BEarly^ NVC pattern than those with an BActive^ or BLate^ pattern (p < 0.001). The BMD values and the T-score measured on the vertebral column, the femoral neck, and the whole femur were significantly lower in SSc and RA patients than those in the control group (p < 0.001) (Table 1). There was no statistically significant difference in the BMD values and T-score of SSc patients whatever the NCV pattern (BLate^ vs BActive^ and BEarly^ NVC patterns) (p > 0.05). The SSc patients had the lowest 25 hydroxyvitamin D serum levels (p < 0.001), with statistically significantly higher serum levels in those SSc patients that had an BEarly^ pattern than in those with either an BActive^ or BLate^ NVC pattern (p = 0.002). Similarly, serum alkaline phosphatase bone levels were significantly lower in those with a BLate^ NVC pattern than those in SSc patients with either an BActive^ or BEarly^ NVC pattern (< 0.0001).
Clin Rheumatol Table 2 Trabecular Bone Score (TBS), bone mineral density (BMD), and bone parameters in SSc patients with different nailfold videocapillaroscopy patterns (NVC patterns: BEarly,^ BActive,^ BLate^)
Early, n = 20
Active, n = 20
Late, n = 20
p value
Age, years (SD) BMI, kg/m2 (SD)
56.4 (7.0)
56.7 (6.9)
57.0 (7.3)
> 0.05
21.3 (2.0)
21.7 (2.1)
21.9 (1.6)
> 0.05
Age of menopause, years (SD) Disease duration, years (SD)
51.7 (2.3) 8.4 (4.7)
51.6 (2.2) 6.1 (4.8)
51.9 (2.3) 11.5 (4.1)
> 0.05 0.05
Raynaud’s duration, years (SD) 25(OH)D, ng/ml (SD) ALP bone, U/I (SD)
9.9 (3.1)
10.2 (2.9)
10.6 (3.0)
0.03
14.1 (7.5) 8.4 (2.3)
12.1 (5.3) 8.2 (1.4)
9.1 (7.1) 6.7 (1.8)
0.002 < 0.0001
Dkk-1, pg/ml (SD)
1827 (888.3)
2290 (487.8)
3467 (954.1)
< 0.0001
TBS, unit-less (SD) Lumbar spine (L1-L4) BMD, g/cm2 (SD) T-score (L1-L4) Femoral neck BMD, g/cm2 (SD)
1.121 (0.56) 0.954 (0.138)
1.104 (0.43) 0.981 (0.128)
0.989 (0.32) 0.977 (0.146)
< 0.0001 > 0.05
− 1.605 (1.267) 0.866 (0.122)
− 1.596 (1.483) 0.799 (0.105)
− 1.611 (1.784) 0.812 (0.166)
> 0.05 > 0.05
− 1.895 (1.245) 0.622 (0.111)
− 1.904 (1.567) 0.598 (0.127)
− 1.881 (1.679) 0.602 (0.108)
> 0.05 > 0.05
− 2.660 (1.586) 0.693 (0.153) − 1.437 (1.442) 0.906 (0.134) − 1.597 (1.349)
− 2.584 (1.539) 0.698 (0.203) − 1.392 (1.863) 0.899 (0.231) − 1.634 (1.538)
− 2.648 (1.347) 0.674 (0.153) − 1.410 (1.541) 0.802 (0.156) − 1.611 (1.245)
> 0.05 > 0.05 > 0.05 > 0.05 > 0.05
T-score (femoral neck) Ward’s triangle BMD, g/cm2 (SD) T-score (Ward’s triangle) Trochanter BMD, g/cm2 (SD) T-score (trochanter) Total hip BMD, g/cm2 (SD) T-score (total hip) ALP bone, bone alkaline phosphatase
There was no significant difference in the levels of PTH, calcium, or phosphorus serum concentrations in SSc (also with different microangiopathy patterns), RA, or CNT, nor was there any correlation between these values and the NVC pattern in SSc patients (p > 0.05). There was no significant difference in the smoking status percentage in SSc (also with different microangiopathy patterns), RA, and controls (p > 0.05). A total of 22/60 SSc patients (37%) reported having had a previous vertebral fracture and all of them had a low bone mass, 12/60 (20%) osteopenia, and 10/60 (17%) osteoporosis. The Dkk-1 serum values were significantly higher in the subgroup of SSc patients with previous vertebral fractures than in those without fractures (3314.0 ± 725.1 vs 2722.1 ± 869.2; p < 0.01). As expected, TBS (0.990 ± 0.081 vs 1.178 ± 0.072; p < 0.0001) and BMD (lumbar spine 0.982 ± 0.164 vs 1.016 ± 0.472; p = 0.006; femoral 0.755 ± 0.132 vs 0.851 ± 0.122; p = 0.009) values were lower in the subgroup of SSc patients with previous vertebral fractures.
Discussion To the best of our knowledge, this is the first time that it has been demonstrated that Dkk-1 serum levels are significantly higher in SSc patients with a BLate^ NVC pattern and are negatively correlated with reduced TBS values. Moreover, the lowest TBS values were observed in the SSc patients with more severe microvascular damage and advanced disease (i.e., the BLate^ NVC
pattern) [15]. Lastly, Dkk-1 levels were shown higher in SSc patients than those in the RA patients and controls. Furthermore, the study confirmed that patients with chronic rheumatic inflammatory diseases, such as RA and SSc, have a higher Dkk-1 level than CNT [8–11]. Dkk-1 is a soluble inhibitor of the canonical Wnt pathway and recent evidence suggests that this molecule regulates several aspects of both bone metabolism and fibrosis [1–5, 8–14, 31, 32]. Data reported in several studies suggest that patients with RA and high circulating Dkk-1 levels have a higher rate of radiographic progression [33]. Although a great deal of interest has been shown in exploring the utility of Dkk-1 as a biomarker in rheumatic diseases, such as RA or AS, more data are necessary before this molecule can be applied to clinical practice [9–11, 33]. Furthermore, a great deal of recent experimental evidence has shown that the initial point of view that Dkk-1 is a bonespecific molecule is incorrect. Indeed, a wide range of tissues, including the skin, have been shown to express Dkk-1 [12]. Data from animal models suggest that Dkk-1 effectively antagonizes Wnt signaling, inducing suppression of mechanisms leading to fibrogenesis in multiple organs [11–14, 32, 33]. Although further confirmation is required, we suggest that all these observations might well support the role of Dkk-1 as a hallmark of bone remodeling. This hypothesis is supported by our finding that there were higher Dkk-1 values in the subgroup of SSc patients with previous vertebral fractures than in those without fractures.
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Interestingly, our SSc patients had a significant correlation between Dkk-1 and 25 hydroxyvitamin D and no significant correlation with PTH, bone alkaline phosphatase, calcium, or phosphorus. Our data is in agreement with that of the World Health Organization, published in 1994, where they confirmed a higher osteoporosis and osteopenia risk in rheumatic patients [34–37]. This study also confirmed that the TBS value, an index of bone texture obtained by the standard BMD values, is lower in SSc and RA patients than that in healthy controls. Furthermore, we confirmed lower 25(OH)D serum levels in SSc patients than in the control group [6, 38–40]. As previously reported, the 25(OH)D value was correlated with TBS but not with BMD, and this observation confirmed that the two techniques evaluate different aspects of bone [15]. The patients with BLate^ NVC pattern showed a significantly lower alkaline phosphatase levels than those with an BActive^ or BEarly^ pattern. This finding is most likely correlated to a reduced bone turnover and bone neoformation [15]. We are aware that the relatively small sample size is a limitation of this study, but this is due to the single-center recruitment and the exclusion criteria. Moreover, almost 85% of the RA cases were treated with low-dose glucocorticoids (≤ 5 mg prednisone/day). In conclusion, the association between bone damage, studied by TBS, DXA and Dkk-1, and the BLate^ NVC pattern of microvascular damage (advanced disease) as well as the correlation between Dkk-1 and the duration of Raynaud’s phenomenon may suggest that diffuse hypoxia/ischemia, related to the diffuse microangiopathy, could be one of the promoting factors for osteoclastogenesis and bone damage/loss in SSc.
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Acknowledgments The authors would like to thank Barbara Wade, contract Professor at the University of Torino, for her linguistic advice.
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Funding statement No specific funding was received from any bodies in the public, commercial, or not-for-profit sectors to carry out the work described in this manuscript. Barbara Ruaro is supported by a Grant from the Italian Society of Rheumatology. Vanessa Smith is a Senior Clinical Investigator of the Research Foundation - Flanders (Belgium) (FWO) [1802915N].
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Compliance with ethical standards Disclosures None. Ethical standards This prospective study was carried out in accordance with the ethical standards stipulated in the 1964 Declaration of Helsinki and its later amendments and was evaluated by the local IRB.
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