Skip Navigation

Rheumatology 2006 45(11):1349-1355; doi:10.1093/rheumatology/kei277
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Davies, C. A.
Right arrow Articles by Herrick, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Davies, C. A.
Right arrow Articles by Herrick, A. L.
Related Collections
Right arrow Systemic Sclerosis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Expression of osteonectin and matrix Gla protein in scleroderma patients with and without calcinosis

C. A. Davies1, M. Jeziorska1, A. J. Freemont1 and A. L. Herrick1,2

1School of Medicine, University of Manchester and 2Rheumatic Diseases Centre, Hope Hospital, Salford, UK.

Correspondence to: A. L. Herrick, Rheumatic Diseases Centre, Clinical Sciences Building, Hope Hospital, Eccles Old Road, Salford M6 8HD, UK. E-mail: ariane.l.herrick{at}manchester.ac.uk


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. Our aim was to evaluate (i) whether the bone matrix proteins osteonectin and matrix gamma-carboxyglutamic acid protein (MGP) are up-regulated in skin biopsies from patients with systemic sclerosis (SSc) and (ii) whether there is differential expression between patients with and without dermal calcinosis, a distressing and debilitating complication of SSc.

Methods. Skin punch biopsies were taken from the forearms of 38 SSc patients with the limited cutaneous subtype of SSc [17 without calcinosis (lcSSc) and 21 with calcinosis (lcSScCal)] and from 11 healthy control subjects. Immunohistochemistry was performed with antibodies to osteonectin and MGP. Staining was assessed semiquantitatively in the microvascular endothelium and in dermal fibroblasts. The Kruskal–Wallis one-way ANOVA was used to compare the data between patient groups.

Results. Both lcSSc and lcSScCal groups showed a statistically significant increase in the percentage of microvessels with osteonectin-positive endothelial cells (EC) (especially the lcSScCal group), whereas lcSScCal alone showed an increase in the percentage of microvessels with MGP-positive EC when compared with controls. In both SSc groups, the percentage of osteonectin and MGP-stained fibroblasts was increased in the reticular dermis (for osteonectin this was more marked in the lcSScCal group). In the papillary dermis, the percentage of osteonectin-stained fibroblasts was increased in both SSc groups but the lcSScCal group alone had a higher percentage of MGP-stained fibroblasts.

Conclusions. When compared with controls, protein expression of osteonectin and MGP was greater in SSc patients generally, and osteonectin expression was significantly higher in EC and fibroblasts of the lcSScCal patients than the lcSSc patients without calcinosis.

KEY WORDS: Osteonectin, Matrix Gla protein, Systemic sclerosis, Immunohistochemistry, Skin.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Very little is known about the pathogenesis of dystrophic calcification in systemic sclerosis (SSc), despite the fact that it is a major clinical problem in a subgroup of patients. It occurs in 20–40% of patients with limited cutaneous disease, typically appearing 10 yr or more after disease onset [1, 2]. In the absence of any apparent disorder in calcium or phosphate metabolism, the question arises as to whether there is a fundamental difference in the biology of skin in SSc patients who develop calcification.

In the last decade some of the studies on vascular calcification have indicated that calcification in atherosclerotic arteries is an active process regulated by inhibitors and activators in a similar way to normal bone formation [3, 4]. In contrast, others have proposed a passive model in which calcification occurs as soon as inhibitors are removed from the matrix [5, 6]. We have shown that calcification in human atherosclerotic plaques sometimes manifests the characteristic features of osteogenesis and takes the form of mineralized bone deposits [7]. Several proteins involved in regulating bone mineralization have been identified both in in vivo and in vitro studies of vascular calcification, including activators such as osteopontin, osteonectin and the signalling molecules bone morphogenetic protein-2 and -4 (BMP-2, BMP-4) and inhibitors such as matrix gamma-carboxyglutamic acid (Gla) protein (MGP), bone sialoprotein and osteocalcin [8–15].

A key issue is whether some of the proteins implicated in vascular calcification and/or bone mineralization might contribute to the pathogenesis of SSc-related calcinosis. Osteonectin, otherwise known as BM-40 or SPARC (secreted protein acidic and rich in cysteine) is a calcium-binding glycoprotein involved in matrix mineralization, wound healing, tissue remodelling and fibrosis [16–19]. In calcification, osteonectin is regarded as a promoter. In bone mineralization, Termine et al. [16] have shown that osteonectin bound to collagen type I forms a complex which can bind to synthetic apatite crystals and free calcium ions and may be involved in both the initial and progressive stages. Osteonectin has been investigated in SSc—elevated osteonectin mRNA levels have been shown in cultured fibroblasts which correlated with increases in type I collagen levels, suggesting involvement in tissue remodelling [20]. Interestingly, circulating concentrations of osteonectin in patients with limited SSc, but not diffuse SSc, have been reported to be significantly higher than in controls [21]. In addition, a study investigating gene expression profiles in SSc patients identified increased gene and protein expression of osteonectin, in cultured dermal fibroblasts [22]. However, two recent investigations on the association between polymorphisms in the osteonectin gene and susceptibility to SSc have yielded conflicting results [22, 23].

Another matrix protein, MGP, was associated with ectopic calcified deposits in skin in an early study of two patients, one with SSc and the other with dermatomyositis [24]. MGP is a vitamin K-dependent calcium-binding protein commonly found in bone [25], cartilage [26] and arterial walls [27] and is currently viewed as a potent inhibitor of calcification [14, 15, 28].

It is not known which cells are involved in the calcification process in SSc, but in vitro and in vivo experiments on vascular calcification have demonstrated a mineralizing phenotype in vascular smooth muscle cells and pericytes associated with the expression of various bone regulatory proteins [15, 29–31]. The purpose of this investigation was to improve our understanding of the calcification process in SSc by locating, at the cellular level, bone regulatory proteins that may be involved. In this initial study we have assessed the expression of the calcification promoter osteonectin and the inhibitor MGP, using immunocytochemistry on skin samples from groups of SSc patients with and without calcinosis.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 Acknowledgements
 References
 
Patients
Full clinical details including disease subtype and duration and autoantibody status were available for all SSc patients (n = 38) and healthy controls (n = 11). Only patients with the limited cutaneous subtype of SSc (lcSSc) were included (skin involvement restricted to distal to the elbows, knees and neck) [32] because calcinosis is more prevalent in patients with limited cutaneous than with diffuse cutaneous disease [1]. Ethical approval was obtained from Salford and Trafford Local Research Ethics Committee and informed, written consent was obtained from all patients. The study was performed on forearm punch biopsies to examine the hypothesis that skin from patients with calcinosis may have an altered matrix composition compared with patients without calcinosis. Areas of calcinosis were not biopsied due to concerns about the risk of poor healing, and also because calcification would interfere with or mask the changes precipitating calcification. The patients were divided into three experimental groups (Table 1): (i) 11 healthy control subjects, (ii) 17 lcSSc patients without calcinosis and (iii) 21 lcSSc patients with calcinosis (lcSScCal). Patients were defined as having calcinosis on the basis of this being evident on examination, or by a convincing history of previous extrusion of calcinotic deposits.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Clinical characteristics of patients and controls

 
Tissue preparation
Skin punch biopsies (4 mm) were taken from the flexor aspect of the forearm. The fresh tissue was immediately placed into 10% neutral buffered formalin, routinely processed and embedded in paraffin wax. Serial sections were cut at 5 µm from each block and mounted on slides coated with APES (3-aminopropyl-triethoxysilane, A-3648; Sigma-Aldrich, Poole, UK). The histological appearance of the skin was graded on two or three haematoxylin and eosin-stained sections from each biopsy by an experienced pathologist (AJF). This yielded a total of 21 grade 0 biopsies (normal skin±minimal perivascular oedema and/or pigmentary incontinence) and 17 grade 1 biopsies (normal appearance of the dermal matrix but with marked perivascular oedema±perivascular mononuclear cell aggregation and/or microvascular basement membrane thickening). Further consecutive sections were used for immunohistochemistry.

Immunolocalization of osteonectin and MGP
Osteonectin protein was detected with a rabbit polyclonal antibody (499255, Calbiochem Novabiochem, Nottingham, UK) that recognizes the 43 kDa non-collagenous osteonectin in human bone. MGP was localized with a mouse monoclonal antibody ({alpha}3–15 IgG, VitaK, Maastricht, The Netherlands) raised against a synthetic peptide corresponding to amino acids 3–15 of non-phosphorylated human MGP. All primary and secondary antibodies were diluted with 0.05 M TRIS-buffered saline (pH 7.6) containing 0.1% bovine serum albumin [TBS/BSA (BSA, A7906, Sigma Chemical Co. Ltd, Gillingham, UK)]. A standard avidin-biotin-peroxidase method was used as follows: endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in 0.05 M TBS for 30 min, then non-specific binding was blocked by incubation in normal goat serum diluted 1/10 in TBS/BSA for 1 h. After overnight incubation at 5°C with the primary antibodies diluted in TBS/BSA (MGP: 1/400; osteonectin: 1/500), sections were incubated with biotinylated goat anti-rabbit (BA 1000, Vector) or goat anti-mouse secondary antibody (sc-2039, Santa-Cruz Biotechnology) diluted 1/500 in TBS/BSA followed by peroxidase conjugated Avidin D (A-2004, Vector) at 1/1000 in TBS, both for 1 h at room temperature. The immunoreactive product was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAB) tablets (D-4168, Sigma). Negative controls consisted of replacing the primary antibodies with TBS/BSA and with rabbit or mouse immunoglobulins (X0903, X0931, DakoCytomation) at an IgG concentration to match that of the primary antibodies. The sections were counterstained with haematoxylin.

Qualitative staining
The overall pattern of immunostaining was examined microscopically in the papillary and reticular dermis of the biopsies.

Quantitative analysis of staining
The staining was evaluated on coded sections using the maximum possible number of non-overlapping fields delineated by an eyepiece graticule (grid square 1 mm, microscope objective x40). The number of fields analysed per section was 11 on average, ranging from 7 to 15. Osteonectin and MGP staining in endothelial cells (EC) associated with microvessels in the papillary dermis and the transition zone between the papillary and reticular dermis was scored as the percentage of microvessels with immunostained cells: score of 1 ≤ 25%, 2 = 25–75% and 3 ≥ 75%. The percentage of immunostained fibroblasts was scored, in the same way, in both papillary and reticular dermis. As there are no markers which allow unequivocal discrimination between pericytes and fibroblasts, pericytes were identified as spindle-shaped cells in close proximity to microvessels and fibroblasts as spindle cells with a clear dissociation/separation from vessels. The intensity of staining for osteonectin and MGP was also assessed in the EC, fibroblasts and extracellular dermal matrix. The staining intensity was scored from 0 (no staining) and 1 (low), 2 (medium) or 3 (intense).

Statistical analysis
The Kruskal–Wallis one-way ANOVA was used to compare group mean ranks followed by homogeneity of variance and post hoc multiple comparison testing to identify any differences.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 Acknowledgements
 References
 
Qualitative analysis of osteonectin and MGP staining
Immunostained product (both osteonectin and MGP) was seen in epidermal keratinocytes, in dermal EC, pericytes and fibroblasts, and on extracellular matrix especially around glandular and vascular structures. The extracellular dermal matrix was more commonly stained for osteonectin than for MGP. In normal skin, cells of all types were usually negative or faintly stained compared with the SSc groups (Fig. 1). Inflammatory cells in both SSc groups were usually intensely stained.


Figure 1
View larger version (101K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1. Immunoperoxidase staining for osteonectin (A–D) and MGP (E–H) in control and SSc skin sections (5 µm) counterstained with haematoxylin. (A) Endothelial cells (arrows) weakly stained for osteonectin in the papillary dermis of control skin. (B) Endothelial cells (arrows) and pericytes intensely stained for osteonectin in the papillary dermis of lcSSc skin. (C) Fibroblasts (arrows) negatively stained for osteonectin in the reticular dermis of control skin. (D) Fibroblasts (arrows) intensely stained for osteonectin in the reticular dermis of lcSScCal skin. (E) Endothelial cells (arrows) negatively stained for MGP in microvessels from control skin. (F) Endothelial cells (arrows), pericytes, fibroblasts and inflammatory cells (arrows in insert) intensely stained for MGP in the papillary dermis of lcSScCal skin. (G) Fibroblasts (arrows) negatively stained for MGP in the reticular dermis of control skin. (H) Fibroblasts (arrows) intensely stained for MGP in the reticular dermis of lcSSc skin. Scale bars: A, B = 80 µm; C, D = 50 µm; E, F = 100 µm; G, H = 50 µm.

 
Quantitative analysis
The data are presented as bar charts in Figs 2–5GoGoGo. Although there were trends towards increased levels in lcSSc compared with controls, and in lcSScCal compared with lcSSc (Figs 2–5GoGoGo), because there was substantial variation within each group some differences did not reach statistical significance (P<0.05).


Figure 2
View larger version (44K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2. Percentage and intensity of osteonectin (ON) and MGP stained microvessels in the papillary dermis of control (C), lcSSc and lcSScCal subjects: (1) ON, *P = 0.024, **P = 0.001, ***P<0.0005 in lcSSc and lcSScCal compared with controls; {blacksquare}, P = 0.025 in lcSScCal compared with lcSSc; (2) MGP, •, P = 0.045 in lcSScCal compared with controls.

 

Figure 3
View larger version (47K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 3. Percentage of osteonectin (ON) and MGP stained fibroblasts in the papillary (Pap) and reticular (Ret) dermis of control (C), lcSSc and lcSScCal subjects: (1) ON, *P = 0.005, **P = 0.001, ***P<0.0005 in lcSSc and lcSScCal compared with controls; {blacksquare}, P = 0.033 in lcSScCal compared with lcSSc; (2) MGP, •, P = 0.033; ••, P = 0.010; •••, P = 0.003 in lcSSc and lcSScCal compared with controls.

 

Figure 4
View larger version (45K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 4. Intensity of osteonectin (ON) and MGP stained fibroblasts in the papillary (Pap) and reticular (Ret) dermis of control (C), lcSSc and lcSScCal subjects: (1) ON, **P = 0.002, ***P<0.0005 in lcSSc and lcSScCal compared with controls; (2) MGP, •, P = 0.014; •••, P<0.0005 in lcSSc and lcSScCal compared with controls.

 

Figure 5
View larger version (48K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 5. Intensity of osteonectin (ON) and MGP stained extracellular matrix in the papillary (Pap) and reticular (Ret) dermis of control (C), lcSSc and lcSScCal subjects. MGP, •, P = 0.029 in lcSScCal compared with controls.

 
Quantitative analysis of osteonectin staining
Endothelial cells
Generally, there were few microvessels present in the deeper reticular dermis, therefore the sampling area was restricted to the papillary dermis including the transition zone between the papillary and reticular dermis. There were significant increases in both SSc groups in the percentage of osteonectin stained microvessels (lcSSc, P = 0.024; lcSScCal, P<0.0005), and in the staining intensity of the EC compared with controls (lcSSc, P = 0.001; lcSScCal, P = 0.001). In addition, lcSScCal had a higher percentage of stained microvessels than lcSSc (P = 0.025), but no significant difference in staining intensity (Fig. 2).

Fibroblasts
In both lcSSc and lcSScCal groups the percentage of immunostained fibroblasts was significantly increased in the papillary (lcSSc, P = 0.001; lcSScCal, P<0.0005) and the reticular dermis (lcSSc, P = 0.005; lcSScCal, P<0.0005) compared with the control group (Fig. 3). Further, the lcSScCal group had an increased percentage of immunostained fibroblasts compared with lcSSc in the reticular dermis only (P = 0.033) (Fig. 3). The staining intensity of the fibroblasts was also increased compared with controls in the papillary (lcSSc, P = 0.002; lcSScCal, P = 0.002) and the reticular dermis (lcSSc, P<0.0005; lcSScCal, P<0.0005), but there were no significant differences between the SSc groups (Fig. 4).

Quantitative analysis of MGP staining
Endothelial cells
In the lcSScCal group only there was a significant increase (P = 0.045) in the percentage of microvessels with immunostained EC compared with controls, but no difference between lcSSc and lcSScCal. There were no significant differences in the staining intensity of the EC (controls compared with lcSScCal, P = 0.086) (Fig. 2).

Fibroblasts
In the lcSScCal group only there was a significant increase in the papillary dermis in the percentage of immunostained fibroblasts (P = 0.010) compared with controls. In the reticular dermis both SSc groups had a higher percentage of stained cells (lcSSc, P = 0.033; lcSScCal, P = 0.003), but there were no significant differences between the lcSSc and lcSScCal groups (Fig. 3). The staining intensity of the fibroblasts was increased compared with controls in the papillary dermis in lcSScCal only (P = 0.014). In the reticular dermis, the staining intensity was higher than controls in both SSc groups (lcSSc, P = 0.014; lcSScCal, P<0.0005), but again there were no significant differences between the SSc groups (Fig. 4).

Osteonectin and MGP staining in the dermal matrix
There were no significant group differences in the intensity of staining in the extracellular matrix, except an increase in MGP staining in the reticular dermis of the lcSScCal group compared with controls (P = 0.029) (Fig. 5).


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
Acknowledgements
 References
 
In the present study we examined the expression of osteonectin and MGP to assess their possible involvement in SSc-associated calcification. Osteonectin and MGP proteins were localized in EC, pericytes, fibroblasts, inflammatory cells and dermal matrix in forearm biopsies of uninvolved/minimally involved skin from lcSSc patients with and without cutaneous calcinosis, and to a lesser extent in controls. The staining pattern of osteonectin agrees with previous findings in normal adult human skin [33, 34]. Also, in association with atherosclerotic calcification osteonectin has been localized in smooth muscle cells, EC and macrophages [10]. MGP expression has not been reported in normal skin but is present in EC, smooth muscle cells, elastic fibres and extracellular matrix in non-diseased human arteries [11, 12].

Our quantitative studies revealed that SSc skin had an increase in the percentage of microvessels with osteonectin-positive EC in both lcSSc groups, which was more marked in the calcinosis group, whereas only the calcinosis group showed a significant increase in the percentage of microvessels with MGP-positive EC when compared with healthy control subjects. Analysis of the percentage of immunostained fibroblasts in the papillary dermis showed an increase in osteonectin and MGP staining in the calcinosis group, but in osteonectin alone in the lcSSc group. In the reticular dermis both groups showed clear increases in the percentage and staining intensity of osteonectin and MGP-positive fibroblasts. Further, osteonectin, but not MGP expression, was significantly increased in EC and fibroblasts in the reticular dermis of calcinosis patients compared with patients without calcinosis.

MGP has commonly been located at sites of calcification in vascular pathologies [11–14] where it has a role as a calcification inhibitor. This is best evidenced in MGP deficiencies which are associated with extensive vascular calcification [28, 35]. In calcified atherosclerotic lesions Dhore et al. [11] reported the up-regulation of the osteonectin gene and protein, but not of MGP. They concluded that vascular calcification is the consequence of sustained levels of inhibitors being suppressed by increased levels of inducers such as osteonectin, BMP-2 and BMP-4. Recently, in vitro and in vivo experiments have shown that MGP binds to BMP-2 to inhibit calcification [3, 14, 15, 36–38]. In order to bind BMP-2, MGP must be in the gamma-carboxylated form which necessitates the presence of vitamin K as a cofactor for the enzyme gamma-glutamyl carboxylase that modifies glutamic acid residues to Gla residues [12]. Significantly, Sweatt et al. [14] have produced evidence from a study of calcified vessels in ageing rats showing that, although MGP levels were raised, the MGP was under-gamma-carboxylated and did not bind BMP-2. Our data derived from SSc skin biopsies revealed an increased expression of MGP. However, the commercially available antibody we used is known to recognize both gamma-carboxylated and under-gamma-carboxylated MGP and if in SSc MGP is mostly under-gamma-carboxylated, this could potentially lead to calcification. In the absence of comparative data from calcinotic SSc tissue it is difficult to explain our findings.

Wallin et al. [37] have proposed that oxidative stress may be a contributory factor to conditions in which arterial calcification is common, and have proposed several mechanisms as to how oxidative stress could prevent vitamin K from carrying out its normal functions, possibly leading to under-gamma-carboxylated MGP. There is substantial evidence, including our own investigations, for the involvement of free-radical-induced oxidative stress in the pathology of SSc [39–44]. In addition, patients with calcinosis have been shown to have an exaggerated microvascular pathology indicated by more telangiectases, giant capillaries and a reduced capillary density than those without calcinosis [45]. Other typical vascular abnormalities are increases in digital infarcts and digital ischaemia. Therefore it seems feasible that MGP in SSc tissue could be largely under-gamma-carboxylated, and especially in sites predisposed/susceptible to calcinosis, the balance between MGP and BMP-2 (and other inhibitors/promoters) levels shifts to favour the latter, resulting in calcification.

In conclusion, we have shown increased expression of two calcification-related proteins in minimally involved forearm skin from patients with lcSSc. This increased expression is more marked in those patients with calcinosis. Our findings signify the need for further research into activators and inhibitors of calcification in skin and subcutaneous tissue of patients with SSc. At present, there is no effective treatment for SSc-related calcinosis. Further elucidation of its pathogenesis is a necessary first step in informing new approaches to therapy.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by the Raynaud's and Scleroderma Association, UK.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Steen VD, Ziegler GL, Rodnan GP, Medsger TA Jr. (1984) Clinical and laboratory associations of anticentromere antibody in patients with progressive systemic sclerosis. Arthritis Rheum 27:125–31.[Web of Science][Medline]
  2. Chamberlain AJ and Walker NPJ. (2003) Successful palliation and significant remission of cutaneous calcinosis in CREST syndrome with carbon dioxide laser. Dermatol Surg 29:968–70.[CrossRef][Web of Science][Medline]
  3. Bostrom K. (2001) Insights into the mechanism of vascular calcification. Am J Cardiol 88:20E–22E.[Web of Science][Medline]
  4. Doherty TM, Asotra K, Fitzpatrick LA, et al. (2003) Calcification in atherosclerosis: bone biology and chronic inflammation at the arterial crossroads. Proc Natl Acad Sci USA 100:11201–6.[Abstract/Free Full Text]
  5. Vermeer C and Braam L. (2001) Role of K vitamins in the regulation of tissue calcification. J Bone Miner Metab 19:201–6.[CrossRef][Web of Science][Medline]
  6. Schinke T and Karsenty G. (2000) Vascular calcification—a passive process in need of inhibitors. Nephrol Dial Transplant 15:1272–4.[Free Full Text]
  7. Jeziorska M, McCollum C, Wooley DE. (1998) Observations on bone formation and remodelling in advanced atherosclerotic lesions of human carotid arteries. Virchows Arch 433:559–65.[CrossRef][Web of Science][Medline]
  8. Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. (1993) Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest 91:1800–9.[Web of Science][Medline]
  9. Shanahan CM, Cary NR, Metcalfe JC, Weissberg PL. (1994) High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J Clin Invest 93:2393–402.[Web of Science][Medline]
  10. Bini A, Mann KG, Kudryk BJ, Schoen FJ. (1999) Noncollagenous bone matrix proteins, calcification, and thrombosis in carotid artery atherosclerosis. Arterioscler Thromb Vasc Biol 19:1852–61.[Abstract/Free Full Text]
  11. Dhore CR, Cleutjens JP, Lutgens E, et al. (2001) Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol 21:1998–2003.[Abstract/Free Full Text]
  12. Spronk HM, Soute BA, Schurgers LJ, et al. (2001) Matrix Gla protein accumulates at the border of regions of calcification and normal tissue in the media of the arterial vessel wall. Biochem Biophys Res Comm 289:485–90.[CrossRef][Web of Science][Medline]
  13. Canfield AE, Farrington C, Dziobon MD, et al. (2002) The involvement of matrix glycoproteins in vascular calcification and fibrosis: an immunohistochemical study. J Pathol 196:228–34.[CrossRef][Web of Science][Medline]
  14. Sweatt A, Sane DC, Hutson SM, Wallin R. (2003) Matrix Gla protein (MGP) and bone morphogenetic protein-2 in aortic calcified lesions of aging rats. J Thromb Haemost 1:178–85.[CrossRef][Web of Science][Medline]
  15. Zebboudj AF, Shin V, Bostrom K. (2003) Matrix GLA protein and BMP-2 regulate osteoinduction in calcifying vascular cells. J Cell Biochem 90:756–65.[CrossRef][Web of Science][Medline]
  16. Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR. (1981) Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26:99–105.[CrossRef][Web of Science][Medline]
  17. Motamed K. (1999) SPARC (osteonectin/BM-40). Int J Biochem Cell Biol 31:1363–6.[CrossRef][Web of Science][Medline]
  18. Yan Q and Sage EH. (1999) SPARC, a matricellular glycoprotein with important biological functions. J Histochem Cytochem 47:1495–506.[Free Full Text]
  19. Brekken RA and Sage EH. (2000) SPARC, a matricellular protein: at the crossroads of cell-matrix. Matrix Biol J Int Soc Matrix Biol 19:569–80.
  20. Vuorio T, Kahari VM, Black C, Vuorio E. (1991) Expression of osteonectin, decorin, and transforming growth factor-beta 1 genes in fibroblasts cultured from patients with systemic sclerosis and morphea. J Rheumatol 18:247–51.[Web of Science][Medline]
  21. Macko RF, Gelber AC, Young BA, et al. (2002) Increased circulating concentrations of the counteradhesive proteins SPARC and thrombospondin-1 in systemic sclerosis (scleroderma). Relationship to platelet and endothelial cell activation. J Rheumatol 29:2565–70.[Abstract/Free Full Text]
  22. Zhou X, Tan FK, Reveille JD, et al. (2002) Association of novel polymorphisms with the expression of SPARC in normal fibroblasts and with susceptibility to scleroderma. Arthritis Rheum 46:2990–9.[CrossRef][Web of Science][Medline]
  23. Lagan AL, Pantelidis P, Renzoni EA, et al. (2005) Single-nucleotide polymorphisms in the SPARC gene are not associated with susceptibility to scleroderma. Rheumatology 44:197–201.[Abstract/Free Full Text]
  24. Lian JB, Skinner M, Glimcher MJ, Gallop P. (1976) The presence of gamma-carboxyglutamic acid in the proteins associated with ectopic calcification. Biochem Biophys Res Commun 73:349–55.[CrossRef][Web of Science][Medline]
  25. Hauschka PV, Lian JB, Cole DE, Gundberg CM. (1989) Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiol Rev 69:990–1047.[Free Full Text]
  26. Loeser R, Carlson CS, Tulli H, Jerome WG, Miller L, Wallin R. (1992) Articular-cartilage matrix gamma-carboxyglutamic acid-containing protein. Characterization and immunolocalization. Biochem J 282:1–6.
  27. Wallin R, Cain D, Sane DC. (1999) Matrix Gla protein synthesis and gamma-carboxylation in the aortic vessel wall and proliferating vascular smooth muscle cells–a cell system which resembles the system in bone cells. Thromb Haemost 82:1764–7.[Web of Science][Medline]
  28. Luo G, Ducy P, McKee MD, et al. (1997) Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386:78–81.[CrossRef][Medline]
  29. Proudfoot D, Skepper JN, Shanahan CM, Weissberg PL. (1998) Calcification of human vascular cells in vitro is correlated with high levels of matrix Gla protein and low levels of osteopontin expression. Arterioscler Thromb Vasc Biol 18:379–88.[Abstract/Free Full Text]
  30. Doherty MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, Canfield AE. (1998) Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res 13:828–38.[CrossRef][Web of Science][Medline]
  31. Canfield AE, Doherty MJ, Kelly V, et al. (2000) Matrix Gla protein is differentially expressed during the deposition of a calcified matrix by vascular pericytes. FEBS Lett 487:267–71.[CrossRef][Web of Science][Medline]
  32. LeRoy EC, Black C, Fleischmajer R, et al. (1988) Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol 15:202–5.[Web of Science][Medline]
  33. Porter PL, Sage EH, Lane TF, Funk SE, Gown AM. (1995) Distribution of SPARC in normal and neoplastic human tissue. J Histochem Cytochem 43:791–800.[Abstract]
  34. Hunzelmann N, Hafner M, Anders S, Krieg T, Nischt R. (1998) BM-40 (osteonectin, SPARC) is expressed both in the epidermal and in the dermal compartment of adult human skin. J Invest Dermatol 110:122–6.[CrossRef][Web of Science][Medline]
  35. Meier M, Weng LP, Alexandrakis E, Ruschoff J, Goeckenjan G. (2001) Tracheobronchial stenosis in Keutel syndrome. Eur Respir J 17:566–9.[Abstract/Free Full Text]
  36. Wallin R, Cain D, Hutson SM, Sane DC, Loeser R. (2000) Modulation of the binding of matrix Gla protein (MGP) to bone morphogenetic protein-2 (BMP-2). Thromb Haemost 84:1039–44.[Web of Science][Medline]
  37. Wallin R, Wajih N, Greenwood GT, Sane DC. (2001) Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy. Med Res Rev 21:274–301.[CrossRef][Web of Science][Medline]
  38. Zebboudj AF, Imura M, Bostrom K. (2002) Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2. J Biol Chem 277:4388–94.[Abstract/Free Full Text]
  39. Lau CS, Bridges AB, Muir A, Scott N, Bancroft A, Belch JJ. (1992) Further evidence of increased polymorphonuclear cell activity in patients with Raynaud's phenomenon. Br J Rheumatol 31:375–80.[Abstract/Free Full Text]
  40. Herrick AL, Rieley F, Schofield D, Hollis S, Braganza JM, Jayson MI. (1994) Micronutrient antioxidant status in patients with primary Raynaud's phenomenon and systemic sclerosis. J Rheumatol 21:1477–83.[Web of Science][Medline]
  41. Bruckdorfer KR, Hillary JB, Bunce T, Vancheeswaran R, Black CM. (1995) Increased susceptibility to oxidation of low-density lipoproteins isolated from patients with systemic sclerosis. Arthritis Rheum 38:1060–7.[Web of Science][Medline]
  42. Stein CM, Tanner SB, Awad JA, Roberts LJ, Morrow JD. (1996) Evidence of free radical-mediated injury (isoprostane overproduction) in scleroderma. Arthritis Rheum 39:1146–50.[Web of Science][Medline]
  43. Denton CP, Bunce TD, Darado MB, et al. (1999) Probucol improves symptoms and reduces lipoprotein oxidation susceptibility in patients with Raynaud's phenomenon. Rheumatology 38:309–15.[Abstract/Free Full Text]
  44. Herrick AL and Matucci-Cerinic M. (2001) The emerging problem of oxidative stress and the role of antioxidants in systemic sclerosis. Clin Exp Rheumatol 19:4–8.[Web of Science][Medline]
  45. Vayssairat M, Hidouche D, Abdoucheli-Baudot N, Gaitz JP. (1998) Clinical significance of subcutaneous calcinosis in patients with systemic sclerosis. Does diltiazem induce its regression? Ann Rheum Dis 57:252–4.[Abstract/Free Full Text]
Submitted 18 July 2005; revised version accepted 28 November 2005.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Rheumatology (Oxford)Home page
C. A. Davies, A. L. Herrick, L. Cordingley, A. J. Freemont, and M. Jeziorska
Expression of advanced glycation end products and their receptor in skin from patients with systemic sclerosis with and without calcinosis
Rheumatology, June 19, 2009; (2009) kep151v1.
[Abstract] [Full Text] [PDF]

Home page
 Rheumatology (Oxford)Home page
M. J. H. van Summeren, W. G. M. Spliet, A. van Royen-Kerkhof, C. Vermeer, M. Lilien, W. Kuis, and L. J. Schurgers
Calcinosis in juvenile dermatomyositis: a possible role for the vitamin K-dependent protein matrix Gla protein
Rheumatology, March 1, 2008; 47(3): 267 - 271.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Davies, C. A.
Right arrow Articles by Herrick, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Davies, C. A.
Right arrow Articles by Herrick, A. L.
Related Collections
Right arrow Systemic Sclerosis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?