QJM Advance Access originally published online on June 13, 2005
QJM 2005 98(7):485-492; doi:10.1093/qjmed/hci078
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N-terminal connective tissue growth factor is a marker of the fibrotic phenotype in scleroderma
From the 1Centre for Rheumatology, Royal Free Hospital and University College School of Medicine, London, UK, and 2FibroGen, South San Francisco, California, USA
Address correspondence to Dr R. Stratton, Centre for Rheumatology, Royal Free Hospital, Pond Street, London NW3 2QG. email: r.stratton{at}rfc.ucl.ac.uk
Received 2 November 2004 and in revised form 23 March 2005
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Background: Over-expression of connective tissue growth factor (CTGF) is a hallmark of fibrotic disease, including scleroderma. CTGF acts with the pro-fibrotic cytokine TGFß to promote sustained fibrotic responses in vivo. Elevated production of CTGF might be responsible for maintenance of the fibrotic phenotype in scleroderma. Assays of CTGF or of its fragments are potential non-invasive measures of the fibrotic response in scleroderma.
Aim: To determine the utility of whole, N-terminal, and C-terminal CTGF as surrogate markers for fibrosis in scleroderma.
Design: Cross-sectional controlled study.
Methods: Plasma was collected prospectively from 47 scleroderma patients (26 diffuse scleroderma, 21 limited scleroderma) and 18 healthy controls. At the same time, dermal interstitial fluid was derived by a suction blister technique from the lesional skin of scleroderma patients, and from the forearm skin of healthy controls. Whole, N-terminal, and C-terminal CTGF were assayed by ELISA, using monoclonal antibodies specific for N- and C-terminal epitopes.
Results: N-terminal cleavage products of CTGF were present at elevated levels in the plasma and dermal interstitial fluid of scleroderma patients, compared to healthy controls. N-terminal CTGF levels in plasma and dermal interstitial fluid correlated with severity of skin disease and (negatively) with disease duration. Whole and C-terminal CTGF levels were low in blister fluid and plasma levels were not elevated in disease.
Discussion: These results support a role for CTGF in scleroderma-associated fibrosis and the utility of N-terminal CTGF as a marker of fibrosis.
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Introduction |
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Scleroderma is a connective tissue disease characterized by the progressive fibrosis of skin and internal organs.1 In general, TGFß is found in pathological fibrosis, and is believed to be critical in the initiation of the fibrotic response, due to its ability to induce expression of matrix genes, and to induce the myofibroblast phenotype.2 In scleroderma, TGFß is over-expressed at the leading edge of early fibrotic lesions, but not in established fibrotic lesions.3 TGFß leads to the induction of another profibrotic factor, connective tissue growth factor (CTGF), which acts downstream and in concert with TGFß to drive the overproduction of collagen matrix.4 CTGF over-expression is a feature of fibrotic disorders, including scleroderma.5
Unlike TGFß, CTGF is elevated in established fibrotic lesions in scleroderma.6 CTGF levels are increased in plasma from scleroderma patients, and scleroderma fibroblasts are characterized in part by expression of CTGF under basal conditions.7–9 Since TGFß and CTGF synergize to result in sustained fibrosis in vivo,10 elevated production of CTGF would be expected to increase and sustain scarring, and help to maintain the progressive fibrosis seen in scleroderma.11,12 Because of these findings, CTGF is considered a potential target for therapies against fibrosis.
CTGF protein has been predicted to contain four structural modules following a 37-amino-acid signal peptide as follows: an insulin-like growth-factor binding domain; a chordin-like cysteine-rich domain; a thrombospondin type 1 repeat; and a carboxy-terminal cysteine knot.13 The first two modules are predicted to comprise an N-terminal domain joined to the C-terminal domain by a linker region.13,14 The cysteine-rich module (module 2) within the N-terminal region of CTGF may be acting by binding other growth factors (such as TGFß) in the extracellular space, and by modifying ligand binding activity and bioavailability.15 Conversely, the C-terminal region mediates heparin and integrin binding properties, and is involved with the ability of CTGF to bind the surface of fibroblasts and to promote fibroblast adhesion.13–15
N and C-terminal CTGF cleavage products are found in biological fluids such as uterine flushings,16 but their biological relevance is not known. CTGF, or endogenous fragments of CTGF, within fibrotic lesions might be surrogate markers of fibrotic disease activity in scleroderma, and be usable as outcome measures in clinical trials or during routine clinical practice.
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Patients
All patients were seen at the Rheumatology clinic of the Royal Free Hospital, a tertiary referral centre for patients with scleroderma. Sequential patients seen for follow-up or as new referrals were asked to participate in the study. Healthy controls were recruited from friends or partners of patients or administrative staff. Local ethical committee approval was obtained from the Royal Free Hospital Ethical Committee. All patients and controls gave written informed consent. Patients were classified as having limited or diffuse scleroderma according to the American Rheumatism Association criteria.17 Skin involvement was quantified using a modified Rodnan skin score technique, which scores a total of 20 areas from 0 to 3 (0, uninvolved; 1, possible involvement; 2, definite involvement; 3, severe involvement) to give total scores ranging from 0 to 60.18
In total, 26 patients with diffuse scleroderma, 21 with limited scleroderma, and 18 healthy controls were enrolled and provided plasma and dermal interstitial fluid for analysis. Clinical and laboratory characteristics of scleroderma patients included in the study are shown in Table 1.
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Dermal interstitial fluid was sampled from the fibrotic forearm skin of patients and from the forearm of healthy controls using a suction blister device (Dermovac, Ventipress), with applied 290–310 mmHg suction for 3–4 h, after which dermal fluid was sampled using a 21-gauge needle and stored at –70°C prior to analysis. Fluid obtained using this technique has the properties of an interstitial fluid sample, and does not arise by exudation of fluid and protein from the vascular space.19 At the same time as blister fluid collection, blood samples were drawn and stored as plasma at –70°C prior to analysis.
Assay for CTGF whole, CTGF N-terminal, and C-terminal fragments
CTGF and the cleavage products were assayed at FibroGen by sandwich ELISA, using monoclonal antibodies against epitopes of C- and N-terminal CTGF as capture and secondary antibody, and recombinant CTGF as standard, as described previously.20
Statistical methods
The CTGF data from plasma and interstitial fluid were positively skewed, i.e. values were heaped to the lower end of the distribution but had a long tail up to higher values, and therefore non-parametric analysis methods were used. Medians and IQRs were used for the expression of summary statistics. The Kruskal-Wallis test was used for a comparison of an outcome between several groups, and the Wilcox rank-sum test for differences between two groups21 (STATsimple 2.0.5 for Mac OS9, Nidus Technologies). p values of <0.05 were taken as statistically significant.
Log(e) transformation of CTGF values was found to normalize these data, and therefore correlation between N-terminal CTGF and disease duration and between N-terminal CTGF and severity of skin disease was sought using log(e)-transformed N-terminal CTGF values. A linear relationship between these variables was sought using the method of least squares, and the linear association between variables was quantified as the correlation coefficient R.21
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Expression of CTGF in the plasma of scleroderma patients and healthy controls
Plasma levels of CTGF and the N- and C-terminal cleavage products are shown in Table 2. These results show no significant elevation of whole CTGF or of C-terminal CTGF in the plasma of scleroderma groups. N-terminal CTGF was present at elevated levels in both limited and diffuse scleroderma groups when compared to controls (Figure 1).
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Expression of CTGF in dermal interstitial fluid of scleroderma patients and controls
Dermal interstitial CTGF and cleavage product levels are shown in Table 3. In general, whole CTGF and C-terminal CTGF were present only at low levels and showed no difference between groups. N-terminal CTGF levels were greatly elevated in scleroderma groups (compared to controls), with the highest levels seen in patients with diffuse scleroderma (Figure 1).
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Correlation between N-terminal CTGF and severity of skin involvement
Because of the great elevation of N-terminal CTGF in diffuse scleroderma, we went on to look for correlation between N-terminal CTGF and scleroderma skin score, a measure of the severity of skin fibrosis. Both plasma and blister fluid levels of N-terminal CTGF showed a positive correlation with skin score (Figure 2) (plasma N-terminal vs. skin score R = 0.43, p = 0.0015, blister fluid N-terminal vs. skin score R = 0.39, p < 0.0001).
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Correlation between N-terminal CTGF and disease duration
We were interested to look for correlation between N-terminal CTGF and disease duration because of the tendency for the fibrotic skin lesions to be active and progressive during the early years of disease, and then to slowly improve over subsequent years. Consistent with this, plasma levels of N-terminal CTGF showed a negative correlation with disease duration (Figure 3) (R = –0.44, p = 0.0033). Blister fluid levels of N-terminal CTGF also showed a negative correlation against disease duration (R = –0.21, p = 0.0082).
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Discussion |
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Scleroderma is a severe life-threatening disorder for which there are no specific therapies aimed at the fibrotic response in skin and internal organs, although progress has been made in treatment of the vascular complications and in suppression of the autoimmune response in the disease.22,23 Present efforts are aimed at understanding the molecular events underlying the sustained fibrotic response in scleroderma, because end-stage fibrosis of internal organs and the intima of blood vessels are the major causes of morbidity and mortality in scleroderma. In keeping with this, we have sought to study the expression of CTGF in scleroderma, because CTGF is involved in wound healing and in the fibrotic response. In biological fluids, endogenous proteases cleave CTGF into fragments.16 We were interested in measuring the relative levels of whole CTGF, N-terminal, and C-terminal cleavage products of CTGF in the dermal interstitial fluid from scleroderma dermal lesions, to gain more insight into the biology of CTGF in fibrosis in humans. Intriguingly, in blister fluid drawn from the fibrotic skin lesions of our scleroderma patients, the N-terminal fragment of CTGF predominated, with relatively little whole or C-terminal CTGF present. N-terminal CTGF levels were elevated in scleroderma blister fluids when compared to healthy controls, and the highest levels were seen in diffuse scleroderma patients. Our interpretation of these findings is that during its involvement in the fibrotic response, in vivo CTGF is cleaved to yield the N-terminal fragment, which is liberated into the extracellular space, while the C-terminal fragment remains cell-membrane-associated, or is internalized or degraded.
This model is consistent with the known properties of CTGF in which the C-fragment mediates integrin binding and heparin-dependent cell adhesion, whereas the N-fragment appears to function as a modifier of other growth factors present within the extracellular space.10,13,14 Our measurements of whole, N-terminal, and C-terminal CTGF in plasma of scleroderma patients are consistent with this model, showing great elevation of the N-terminal fragment when compared to control values, but no significant elevation of whole or C-terminal fragment CTGF. These observations support the notion that CTGF has a role in the fibrotic response in scleroderma, and confirm CTGF as a potential target for therapies against scleroderma fibrosis.5,6
For clinicians, an assay for the activity of the fibrotic response would be a useful adjunct to clinical and radiological assessment of organ fibrosis. Measurement of N-terminal CTGF in blister fluid or plasma might be useful clinically, as the levels appear to correlate with severity of skin fibrosis. Also, we found a negative correlation between N-terminal CTGF levels and disease duration, consistent with the natural history of scleroderma showing regression of skin fibrosis after the first 2–3 years of disease.24 A longitudinal study of CTGF N-terminal fragment with disease progression needs to be performed to evaluate more precisely the diagnostic and prognostic utility of this marker.
In conclusion, our data support the involvement of the fibrosis-enhancing growth factor CTGF in scleroderma, showing elevated levels of the N-terminal cleavage product in both extracellular fluid drawn from the fibrotic lesions and in plasma. N-terminal CTGF predominates over whole and C-terminal CTGF in interstitial fluid from the fibrotic skin lesions of scleroderma patients, consistent with the release of N-terminal into the extracellular space following the involvement of CTGF in the fibrotic response. Assay for N-terminal CTGF might be useful as a surrogate marker for the fibrotic response.
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Acknowledgments |
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This study was funded in part by a grant from the Scleroderma Society UK, and we acknowledge their kind support.
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References |
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