Rheumatology Advance Access originally published online on November 8, 2005
Rheumatology 2006 45(3):303-307; doi:10.1093/rheumatology/kei143
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Clinical significance of serum levels of matrix metalloproteinase-13 in patients with systemic sclerosis
Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo and 1 Department of Dermatology and Plastic and Reconstructive Surgery, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
Correspondence to: H. Ihn, Department of Dermatology and Plastic and Reconstructive Surgery, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1–1–1 Honjo, Kumamoto-city, Kumamoto 860-8556, Japan. E-mail: ihn-der{at}kaiju.medic.kumamoto-u.ac.jp
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Abstract |
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Objectives. To investigate the clinical significance of serum matrix metalloproteinase-13 (MMP-13) levels in patients with systemic sclerosis (SSc).
Methods. Serum MMP-13 levels were determined by using a peptide substrate cleavage assay in 20 patients with diffuse cutaneous SSc (dcSSc), 20 with limited cutaneous SSc (lcSSc) and 10 normal controls.
Results. The serum MMP-13 levels in patients with dcSSc or lcSSc were significantly lower than those in normal controls (53.4 ± 14.1 vs 73.2 ± 11.5 ng/ml, P<0.0005; 59.4 ± 14.8 vs 73.2 ± 11.5 ng/ml, P<0.005, respectively), but there was no significant difference in the serum MMP-13 levels between patients with dcSSc and those with lcSSc. Disease duration prior to the diagnosis was significantly shorter in SSc patients with decreased serum MMP-13 levels than in those with normal levels (3.0 ± 2.2 vs 8.6 ± 7.6 yr, P<0.0005). In addition, serum MMP-13 levels were moderately correlated with the duration of the disease (r = 0.451, P<0.05). Though there was no significant difference in the frequencies of pulmonary fibrosis or reduced %DLco (diffusing capacity of lung for carbon monoxide), the frequency of reduced %VC (vital capacity) was significantly greater in patients with decreased serum MMP-13 levels than in those with normal levels (73 vs 24%, P<0.05).
Conclusions. Matrix metalloproteinase-13 may be involved in the fibrotic process of SSc, especially in the initiation of fibrosis. The serum MMP-13 levels may serve as a useful marker for the severity of pulmonary fibrosis in patients with SSc.
KEY WORDS: Disease severity, Disease duration, Pulmonary fibrosis, %VC
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Introduction |
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Systemic sclerosis or scleroderma (SSc) is an acquired disorder which typically results in fibrosis of the skin and internal organs [1]. The fibrosis in this disorder is characterized by uncontrolled excessive deposition of type I collagen, which is thought to be attributed to an imbalance between production and degradation of the protein. Such a process is achieved by coordinating the expression of several functionally distinct but biologically related gene products. In fact, SSc fibroblasts produce an excessive amount of type I collagen [2, 3]. In addition, SSc fibroblasts show a marked decrease in the activity and production of matrix metalloproteinase (MMP)-1 [4], while the expression of tissue inhibitor of metalloproteinase-1, a MMP-1 inhibitor, is increased [5].
Matrix metalloproteinases are important enzymes involved in the breakdown of various extracellular matrix (ECM) components. Matrix metalloproteinases can be divided into subgroups, including collagenases, stromelysins, stromelysins-like MMPs, gelatinases, membrane-type MMPs and others. Matrix metalloproteinase-13 is a member of the collagenase family, which degrades fibrillar collagens of types I, II, III, IV, X and XIV, tenascin, fibronectin, aggrecan, versican and fibrillin-1 [6]. It is now accepted that MMP-13 plays a key role in the MMP activation cascade, both activating and being activated by several MMPs. The expression of MMP-13 has been well studied in cancer and arthritic diseases. Elevated expression of MMP-13 has been documented in numerous malignancies, including adenocarcinoma, squamous cell carcinoma and basal cell carcinoma, and its association with tumour behaviour and patient prognosis has been reported [6]. Significant expression of MMP-13 is observed in highly invasive tumours, suggesting that MMP-13 probably plays a role in regulating tumour invasion, which needs remodelling of ECM. Matrix metalloproteinase-13 is of special interest for the pathogenesis of rheumatoid arthritis (RA) because it cleaves type II collagen of hyaline cartilage more efficiently than two other human collagenases, interstitial collagenase (MMP-1) and neutrophil collagenase (MMP-8) [7]. Significant expression of MMP-13 is demonstrated by immunohistochemistry and in situ hybridization in fibroblast-like cells of the synovial membrane in RA [7–9]. Furthermore, production and activity of MMP-13 in synovial fluid and serum are significantly elevated in patients with RA [10]. These previous data indicate that MMP-13 functions as a useful marker for the activity of diseases characterized by remodelling of the ECM. To our knowledge, however, there have been no reports regarding the role of MMP-13 in the pathogenesis of SSc.
The purpose of this study is to investigate the serum levels of MMP-13 in SSc patients and assess their association with clinical symptoms in this disease.
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Methods |
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Patients
Serum samples were obtained from 40 SSc patients (35 women, 5 men; mean age 50.0 ± 12.0 yr; mean disease duration 7.0 ± 7.1 yr) first evaluated in our division during 1990–2000. Patients who had been treated with corticosteroids or immunosuppressives were excluded. Patients were grouped according to the classification system proposed by LeRoy et al. [11]: 20 patients with limited cutaneous SSc (lcSSc) and 20 patients with diffuse cutaneous SSc (dcSSc). All patients with dcSSc and 19 patients with lcSSc fulfilled the criteria proposed by the American College of Rheumatology [12]. An lcSSc patient who did not meet these criteria had sclerodactyly and at least two other features of the CREST syndrome (calcinosis, Raynaud's phenomenon, oesophageal dysmotility, telangiectasia). As normal controls, 10 serum samples were also obtained from healthy controls (nine women, one man; mean age 51.5 ± 10.2 yr). Informed consent was obtained from all subjects. Aliquots of sera were frozen at –80°C until assayed. The protocol was approved by Graduate School of Medicine, University of Tokyo and The University of Tokyo Hospital.
Clinical assessment
The clinical and laboratory data reported in this study were obtained at the time the blood samples were drawn. Patients were evaluated for the presence of gastrointestinal, pulmonary, cardiac, renal or joint involvement, as described previously [13]. Disease onset was defined as the first clinical event that is a clear manifestation of SSc other than Raynaud's phenomenon. The duration of disease was defined as the interval between the onset and the first physician diagnosis.
Antinuclear antibodies
Antinuclear antibodies (ANA) were detected by indirect immunofluorescence using HEp-2 cells as the substrate and double immunodiffusion.
The measurement of serum MMP-13 levels
According to the manufacturer's instructions, specific kits were used for the measurement of serum MMP-13 levels (Amersham Pharmacia Biotech). In brief, polystyrene cups coated with F(ab')2 goat antimouse were incubated with mouse anti-MMP-13 antibodies at 37°C for 2 h and subsequently incubated with 100 µl of 10-fold diluted serum at 4°C for 24 h. Then, the cups were washed and incubated at 37°C for 1 h with 50 µl of 0.5 mM p-aminophenylmercuric acetate solution, which changes pro-MMP-13 into its active form. Next, the detection reagent, which includes a pro detection enzyme and a specific chromogenic peptide substrate, was added and the absorbance at 405 nm was immediately measured. Then, the cups were incubated at 37°C for 2 h, and the absorbance at 405 nm was measured again. A standard curve was generated by plotting 
Absorbance405 (y-axis) against ng/ml standard (x-axis) and serum MMP-13 levels were calculated from Absorbance405 of each sample using this standard curve. Serum MMP-13 levels more than 2 S.D. lower than the mean level in the normal controls were regarded as decreased.
Statistical analysis
Statistical analysis was carried out with a Student's t-test for the comparison of means and Fisher's exact probability test for the analysis of frequency. Correlations with clinical data were assessed by Spearman's rank correlation coefficient. Statistical significance was defined as a P value of less than 0.05.
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Results |
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Serum levels of MMP-13
The serum MMP-13 levels were significantly lower in SSc patients than in healthy controls (56.4 ± 14.6 vs 73.2 ± 11.5 ng/ml, P < 0.001). The serum MMP-13 levels of dcSSc or lcSSc patients were also significantly lower than those of normal controls (53.4 ± 14.1 vs 73.2 ± 11.5 ng/ml, P < 0.0005; 59.4 ± 14.8 vs 73.2 ±11.5 ng/ml; P < 0.005, respectively). The serum MMP-13 levels in dcSSc patients were decreased compared with those in lcSSc patients, but there was no significant difference. The cut-off value (mean – 2 S.D.) was set at 50.2 ng/ml, based on the data of the 10 healthy control sera. As shown in Fig. 1, decreased serum MMP-13 levels were found in 7 of 20 (35%) dcSSc patients and 4 of 20 (20%) lcSSc patients.
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Correlation of serum MMP-13 levels with clinical and serological features of patients with SSc
The clinical and serological features of patients with decreased or normal levels of MMP-13 are shown in Table 1. There was no significant difference between these groups in terms of sex or age of onset. Disease duration prior to the diagnosis was significantly shorter in patients with decreased serum MMP-13 levels than in those with normal levels (3.0 ± 2.2 vs 8.6 ± 7.6 yr, P < 0.0005). There was no significant difference in the frequency of dcSSc between these two groups. Though there was no significant difference in the frequencies of pulmonary fibrosis (PF) or reduced %DLco (diffusing capacity of lung for carbon monoxide) (73 vs 48% and 81 vs 59%, respectively), the frequency of reduced %VC (vital capacity) was significantly greater in patients with decreased serum MMP-13 levels than in those with normal levels (73 vs 24%, P<0.05).
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Regarding joint involvement, there was no significant difference in the prevalence between these two groups (27 vs 28%). Furthermore, there was no significant difference in serum MMP-13 levels between SSc patients with joint involvement and those without (52.3 ± 17.3 vs 58.3 ± 14.4 ng/ml).
Since the disease duration was significantly shorter in SSc patients with decreased serum MMP-13 levels, we also investigated whether serum MMP-13 levels are associated with disease duration. As expected, serum MMP-13 levels were moderately correlated with disease duration in SSc patients (r = 0.451, P<0.05).
We finally investigated the association of serum MMP-13 levels with the prevalence of decreased %VC in SSc patients with PF. In 11 patients with decreased serum MMP-13 levels, eight patients had PF and all of them showed decreased %VC. By contrast, in 29 patients with normal levels, 14 patients had PF and seven of them showed decreased %VC. The prevalence of decreased %VC in SSc patients with PF was significantly higher in SSc patients with decreased serum MMP-13 levels than those with normal levels (100 vs 50%, P<0.05).
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Discussion |
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The function of MMP-13 has been well-studied in human fetal skin fibroblasts and human gingival fibroblasts. In these cells, the expression of MMP-13 is increased by transforming growth factor beta (TGF-ß), a growth factor implicated in accumulation of ECM in wound repair and fibrosis [14, 15]. It is therefore possible that MMP-13 may play an important role in rapid turnover of collagenous ECM of granulation tissue during normal repair of fetal and gingival wounds, resulting in minimal scar formation. By contrast, TGF-ß showed no significant effect on the expression of MMP-13 in human neonatal skin fibroblasts [15]. However, other reports demonstrated that the expression of MMP-13 was induced by three-dimensional culture in human neonatal skin fibroblasts [16] and the elevated expression of MMP-13 was observed by in situ hybridization in fibroblast- and macrophage-like cells in fibrotic areas of chronic dermal wounds [16]. Although further studies are required, these previous data indicate that MMP-13 may be implicated in the chronic wound healing process.
Considering that MMP-13 is a collagenolytic enzyme with a wide substrate specificity, the present observation that serum levels of MMP-13 are significantly decreased in SSc patients suggests that the decreased expression of MMP-13 contributes to the establishment of fibrosis through reducing the degradation of ECM proteins. Furthermore, disease duration prior to the diagnosis was significantly shorter in SSc patients with decreased serum MMP-13 levels than in those with normal levels, and serum MMP-13 levels were moderately correlated with disease duration. Collectively, these results suggest that the decreased MMP-13 is involved in the initiation of the fibrotic process in SSc.
In general, the severity of PF is evaluated by pulmonary function test. In the normal course of PF, %DLco is initially decreased, which may even precede the onset of symptoms, and subsequent reduction of %VC is observed with disease progression. In this sense, the values of %VC may reflect the severity of PF. Considering this point, the finding that the prevalence of decreased %VC in SSc patients with PF is significantly higher in SSc patients with decreased serum MMP-13 levels than those with normal levels suggests that serum MMP-13 levels reflect the severity of PF in SSc. Since PF is the most frequent respiratory manifestation and is one of the major causes of mortality in SSc, PF is the most important factor which determines the severity of SSc. Thus, serum MMP-13 levels may reflect the disease severity of SSc.
Increasing evidence suggests the involvement of MMP-13 in the pathogenesis of PF. Papakonstantinou et al. [17] demonstrated that normal human lung fibroblasts produced detectable levels of MMP-13 in vitro. Selman et al. [18] showed that the expression of MMP-13 was not detected in lung tissue of people with human idiopathic PF. Furthermore, Ruiz et al. [19] elucidated that the expression levels of MMP-13 were decreased in lung tissue of bleomycin-induced PF compared with normal lung tissue in rat animal models. Taken together with these previous findings, the present observation indicates that MMP-13 may be involved in the development of PF in SSc.
To date, MMP-13 has been well implicated in the pathogenesis of RA. Emerging studies showed that MMP-13 was detected in the synovial membrane in RA but not in normal synovial membrane [7–9]. Furthermore, serum levels of MMP-13 were significantly elevated in patients with RA compared with normal controls [10]. Since polyarthralgia and/or polyarthritis is often observed in SSc, we divided SSc patients into two groups with or without joint involvement and investigated further. However, there was no significant difference in serum MMP-13 levels between SSc patients with joint involvement and those without. Since SSc patients with RA are not included in this study, these data indicate that serum MMP-13 levels have no correlation with joint involvement in SSc. This notion is supported by the difference in the pathogenesis of joint involvement in SSc and RA [20].
In summary, the present study demonstrates that MMP-13 may be involved in the fibrotic process of SSc, especially in the initiation of fibrosis, and the serum MMP-13 levels may serve as a useful marker for the severity of PF in SSc patients. Further studies are required to clarify the significance of MMP-13 in the pathogenesis of SSc.
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The authors have declared no conflicts of interest.
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