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Clin Exp Immunol. Nov 2006; 146(2): 362–370.
PMCID: PMC1942060

Hypoxia induces expression of connective tissue growth factor in scleroderma skin fibroblasts

Abstract

Connective tissue growth factor (CTGF) plays a role in the fibrotic process of systemic sclerosis (SSc). Because hypoxia is associated with fibrosis in several profibrogenic conditions, we investigated whether CTGF expression in SSc fibroblasts is regulated by hypoxia. Dermal fibroblasts from patients with SSc and healthy controls were cultured in the presence of hypoxia or cobalt chloride (CoCl2), a chemical inducer of hypoxia-inducible factor (HIF)-1α. Expression of CTGF was evaluated by Northern and Western blot analyses. Dermal fibroblasts exposed to hypoxia (1% O2) or CoCl2 (1–100 µM) enhanced expression of CTGF mRNA. Skin fibroblasts transfected with HIF-1α showed the increased levels of CTGF protein and mRNA, as well as nuclear staining of HIF-1α, which was enhanced further by treatment of CoCl2. Simultaneous treatment of CoCl2 and transforming growth factor (TGF)-β additively increased CTGF mRNA in dermal fibroblasts. Interferon-γ inhibited the TGF-β-induced CTGF mRNA expression dose-dependently in dermal fibroblasts, but they failed to hamper the CoCl2-induced CTGF mRNA expression. In addition, CoCl2 treatment increased nuclear factor (NF)-κB binding activity for CTGF mRNA, while decreasing IκBα expression in dermal fibroblasts. Our data suggest that hypoxia, caused possibly by microvascular alterations, up-regulates CTGF expression through the activation of HIF-1α in dermal fibroblasts of SSc patients, and thereby contributes to the progression of skin fibrosis.

Keywords: CTGF, dermal fibroblasts, HIF-1α, hypoxia, systemic sclerosis

Introduction

Systemic sclerosis (SSc) is characterized by microvascular alterations and excessive fibrosis of skin and internal organs [1]. Overproduction of extracellular matrix proteins by abnormally activated fibroblasts causes gradual sclerosis of the skin and internal organs such as the lung, kidney and oesophagus [2]. Although the pathogenesis of SSc is not understood completely, transforming growth factor (TGF)-β is essential in this process. In the skin of scleroderma patients, TGF-β expression is elevated compared to healthy controls [35]. TGF-β is a potent inducer of extracellular matrix protein, including collagen and proteoglycan [68], and plays a key role in mediating fibrosis. However, although TGF-β is found in early inflammatory skin lesions, it is rarely detected in advanced fibrotic skin lesions [9]. In addition, fibroblasts of scleroderma patients have decreased responsiveness to TGF-β[10]. Therefore, other factors besides TGF-β may be involved in developing fibrosis.

Connective tissue growth factor (CTGF) is a cysteine-rich fibrogenic cytokine expressed in the endothelium, fibroblasts, chondrocytes and smooth muscle cells, and is induced by stimulation with TGF-β. CTGF promotes the proliferation of the endothelium and fibroblasts [11,12], and has been implicated in diverse fibrotic diseases such as liver cirrhosis, pulmonary fibrosis, renal fibrosis and cardiac fibrosis [1317]. Serum CTGF level is markedly elevated in SSc patients compared to normal people or patients with other autoimmune diseases, and correlates closely with the degree of skin fibrosis [18]. Moreover, CTGF expression is elevated in dermal fibroblasts of SSc patients [19]. When human dermal fibroblast cell lines are transfected by adenovirus-expressing CTGF, collagen synthesis is increased [20]. Conversely, collagen synthesis is decreased when CTGF expression is inhibited by anti-sense mRNA in dermal fibroblasts pretreated with TGF-β[12]. Therefore, CTGF is a critical mediator of TGF-β-induced skin fibrosis in SSc.

In SSc patients vascular changes precede skin sclerosis by several years, and digital oedema, telangiectasis and digital ulcers may develop as disease progresses [21,22]. Vascular changes in SSc are characterized by the loss and structural changes in capillaries. Gradual decreases in the diameter and number of vessels results in a chronically hypoxic state in various organs as well as skin of SSc patients [2125]. Hypoxia-inducible factor-1α (HIF-1α) is a nuclear factor whose DNA binding activity is induced by hypoxia [26]. HIF-1α expression is elevated in a number of fibrotic diseases [2732], suggesting that it may be linked to excessive fibrosis. Moreover, HIF-1α is a critical mediator of TGF-β and CTGF expression in some types of cells [33,34]. However, the association of hypoxia with skin fibrosis and CTGF expression in SSc remains to be defined.

In this study, we demonstrate that dermal fibroblasts stimulated with hypoxia showed increased CTGF expression via activation of HIF-1α. In addition, TGF-β had an additive effect on the hypoxia-induced CTGF expression in dermal fibroblasts. Nuclear factor (NF)-κB was the major pathway responsible for the hypoxia-induced CTGF expression. Our data suggest that chronic hypoxia in SSc instigates CTGF expression through the activation of HIF-1α in dermal fibroblasts, and thereby contributes to the progression of skin fibrosis in SSc.

Materials and methods

Cell culture and hypoxic condition

Dermal fibroblasts were obtained from affected skin biopsies of nine SSc patients and eight healthy controls using a standard explant method. Fibroblasts were grown from explants in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco brl, Grand Island, NY, USA), 2 mM glutamine, penicillin (100 U/ml) and streptomycin (100 µg/ml) and plated in a 60-mm culture dish. The cultures were kept at 37°C in 5% CO2 and the culture medium was replaced every 3 days. When cells approached confluence, they were detached with trypsin-ethylenediamine tetraacetic acid (EDTA), passed after dilution 1:3 with fresh medium, and recultured until used. Cells were used at the third or fifth passage for all experiments. For hypoxic experimentation, dermal fibroblasts grown to 50–80% confluence were either cultured in a hypoxic incubator (Thermo Electron Corp., Marietta, GA, USA) containing 1% O2/5% CO2/10% H2/84% N2, or exposed to various concentrations of CoCl2 (1–100 µM) in a 5% CO2-95% air incubator.

Northern blot analysis

Dermal fibroblasts (5 × 105 cells/100 mm culture dish) were incubated with hypoxic stimuli, and then total RNA (3 µg) was electrophoresed through a 1% agarose gel containing 37% formaldehyde and transferred onto a Hybond-N membrane (Amersham Corp., Amersham, Bucks, UK). The RNA was hybridized to a biotin-labelled human CTGF cDNA and to a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. The biotin-labelled CTGF probe was generated with a Detector Random Primer DNA Biotinlyation kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) using biotin-N4-dCTP. Prehybridization and hybridization were performed at 42°C for the CTGF and GAPDH probes. After hybridization, the membranes were washed and subjected to AP–streptavidin and CDP–Star detection systems using RNA Detector Northern Blotting Kits (Kirkegaard & Perry Laboratories). This chemiluminescent detection system relies on the use of AP, a reporter enzyme that catalyses CDP-Star, a 1,2-dioxetane chemiluminescent substrate, during the last step of the detection system to produce luminescent signals. The chemiluminescent signal was detected by exposing to Fuji X-ray film (Fuji Co., Tokyo, Japan).

Western blot analysis for HIF-1α and CTGF

Dermal fibroblasts were incubated for various times in the presence or absence of CoCl2. After incubation, the cells were harvested and lysed in 50 µl lysis buffer [20 mM Tris-HCl, pH 7·5; 0·5 M NaCl; 1 mM EDTA; 1 mM ethylene glycol tetraacetic acid (EGTA); 0·25% Triton X-100; protease inhibitor cocktail; 2 mM phenylmethylsulphonic fluoride (PMSF); 1 mM dithiothreitol (DTT)] and the resulting lysate was cleared by centrifugation. Protein concentrations were then determined with the Bradford method (Bio-Rad, Hercules, CA, USA). Fifty µg of proteins were resolved on 12% sodium dodecyl sulphide–polyacrylamide electrophoresis gel (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked by overnight incubation at 4°C with phosphate-buffered saline (PBS) containing 5% skimmed milk and 0·1% Tween-20, and then incubated for 2 h at room temperature with a rabbit anti-human HIF-1α antibody (1 µg/ml) (Santa Cruz Biotechnology, CA, USA), rabbit anti-human IκBα antibody (0·5 µg/ml) (Santa Cruz Biotechnology) or anti-human CTGF antibody (2 µg/ml) (R&D Systems, Minneapolis, MN, USA) in the blocking buffer. The membrane was incubated subsequently with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2500 dilution) and HRP-conjugated anti-mouse IgG (1:5000) visualized by chemiluminescence system (ECL, Amersham, Freiburg, Germany). Fuji X-ray film (Fuji Co.) with cassette closure times of 1–30 min was used to obtain adequate exposure and to visualize bands.

Plasmid constructs

The human HIF-1α gene was amplified from cDNA of dermal fibroblasts from scleroderma patients by polymerase chain reaction (PCR) with two primers (1991–2472). The forward primer has a XhoI site and the reverse primer has a BamHI site for cloning. The resulting PCR product was cloned into the enhanced green fluorescent protein (GFP) expression vector (Becton Dickinson, Mountain View, CA, USA), pGFP-C1, to generate the pGFP-HIF-1α construct and verified by sequencing (abi prism® 310, Applied Biosystems, Foster City, CA, USA).

Transfection of HIF-1α gene into human dermal fibroblast cell lines

Because transient transfection of primary isolated skin fibroblasts remained largely inefficient, we used the Detroit 551 cell line, dermal fibroblasts immortalized with SV40 T antigen in our experiments. Plasmid DNA transfection of the HIF-1α gene into the Detroit 551 cell line was performed in six-well plates using LipofectAMINETM 2000 reagent following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The day before transfection, 3 × 104 Detroit 551 cells were plated in 2 ml of medium/well. For each well, LipofectAMINETM reagent (1·6 µl) was mixed with pGFP-C1 vector or pGFP-HIF-1α (0·8 µg) in serum-free Opti-MEM to allow DNA-LipofectAMINE reagent complexes to form. The complexes were added to respective wells and mixed by rocking the plate gently back and forth. After 4 h, the cells were incubated in a CO2 incubator for 24 h in DMEM supplemented with 10% fetal calf serum (FCS). To assay the expression of GFP-HIF-1α, the fluorescence signal was photographed with a laser scanning confocal imaging system (Bio-Rad) 24 h after transfection. The GFP-positive cells were quantified and indicated a transfection efficiency of 50%. Transfected cells were exposed to 100 µM CoCl2 for 4 h, and subjected to indirect immunocytochemistry using an anti-GFP antibody and Northern blot for CTGF mRNA expression. To quantify HIF-1α expression, whole cell extracts were prepared in lysis buffer, and protein concentrations in the extracts were determined by Bradford protein assay (Bio-Rad). Fifty µg of extracts were resolved on a 10% SDS-PAGE gel.

Electrophoretic mobility shift assay (EMSA)

The nuclear extract was harvested from dermal fibroblasts and prepared as described [35]. Oligonucleotide probes containing the NF-κB binding site of the human CTGF promoter (5′-GAG GAA TGT CCC TGT TTG-3′) were generated by 5′ end-labelling of the sense strand with [γ-32P] dATP (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and T4 polynucleotide kinase (Takara Shuzo Co., Shiga, Japan). Unincorporated nucleotides were removed with Micro Bio-Spin(R) 30 chromatography columns (Bio-Rad), and then nuclear extracts (2–5 µg protein) were incubated with radiolabelled DNA probes [300 000–500 000 counts per minute (cpm)] for 30 min at room temperature in a final volume of 20 µl containing 5 × gel shift binding buffer (Promega Corporation, Madison, WI, USA) and 50 ng of poly(dI-dC)/l. Samples were electrophoresed on non-denaturing 6% polyacrylamide gels in 0·5× Tris–borate–EDTA buffer (pH 8·0) at 180 V. Gels were dried under vacuum and exposed to Fuji X-ray film at −70°C for 48 h.

Results

Hypoxia induces HIF-1α expression in dermal fibroblasts

We investigated the expression of HIF-1α protein in dermal fibroblasts in response to treatment of CoCl2. In untreated dermal fibroblasts, the expression of HIF-1α protein was very faint in dermal fibroblasts of normal controls. As shown in Fig. 1, treatment with 100 µM of CoCl2 significantly increased the expression of HIF-1α protein in dermal fibroblasts of normal and sclerodema patients. The expression of HIF-1α protein was seen as early as 30 min, and increased up to 2 h. Induction of HIF-1α protein by CoCl2 persisted up to 8 h. Similar patterns of increase were also detected when cells were cultured in 1% hypoxic incubator (data not shown).

Fig. 1
Expression of hypoxia-inducible factor-1α (HIF-1α) during hypoxia in dermal fibroblasts of normal controls and systemic sclerosis patients. Dermal fibroblasts of normal control (NDF) and systemic sclerosis patients (SSDF) were exposed ...

Induction of CTGF mRNA by hypoxic stimulation in dermal fibroblasts

To determine whether hypoxia enhances the CTGF mRNA expression, dermal fibroblasts were exposed to hypoxic stimuli, such as 1% O2 and 100 µM of CoCl2, a chemical inducer of HIF-1α. As shown in Fig. 2, CTGF mRNA expression in the dermal fibroblasts of SSc patients and normal controls was up-regulated by exposure to hypoxic stimuli. This increase was observed at 0·5 h and peaked at 2 h. Such an increase of CTGF mRNA expression was also observed in Detroit 551 skin fibroblast cell lines when exposed to above hypoxic stimuli (data not shown). These results suggest that exposure to hypoxia may lead dermal fibroblasts to express CTGF constitutively in SSc patients.

Fig. 2
Hypoxic stimuli induce connective tissue growth factor (CTGF) mRNA expression in dermal fibroblasts. Dermal fibroblasts of normal controls and systemic sclerosis patients were exposed to hypoxia (1% O2) and 100 µM of CoCl2 for indicated times. ...

Hypoxia-induced CTGF expression is mediated by HIF-1α

To determine whether the up-regulation of hypoxia-induced CTGF expression is mediated by HIF-1α, the HIF-1α gene was overexpressed in dermal fibroblasts and CTGF expression was then determined in these cells under hypoxic conditions. As shown in Fig. 3a,b, the CTGF mRNA expression was also increased markedly in cells transfected with the GFP-HIF-1α gene compared with untransfected cells or cells transfected with GFP gene only, indicating that HIF-1α is involved in the induction of CTGF mRNA. HIF-1α is translocated from the cytosol to the nucleus when activated by hypoxic stimuli [36]. In our experiment, GFP staining was present in the nucleus and the cytoplasm of GFP-HIF-1α-transfected cells. Upon exposure to CoCl2 (100 µM), GFP staining moved exclusively to the nucleus in GFP-HIF-1α-transfected cells, but not in GFP-only transfected cells (Fig. 3c), suggesting that the HIF-1α activation by hypoxia may elicit the CTGF mRNA transcription. Together, these data indicate that CTGF mRNA transcription in dermal fibroblasts is regulated by HIF-1α triggered by hypoxic stimulation.

Fig. 3
Hypoxia-inducible factor-1α (HIF-1α) mediates the increase in connective tissue growth factor (CTGF) mRNA by hypoxia in scleroderma skin fibroblasts. (a,b) Expression levels of CTGF mRNA (a) and protein (b) in HIF-1α-overexpressed ...

TGF-β additively increases hypoxia-induced CTGF mRNA expression

In an animal model of skin fibrosis, TGF-β transiently induced subcutaneous fibrosis, and serial injections of CTGF after TGF-β caused persistent fibrosis [37]. Therefore, TGF-β may play a role in the initiation of fibrosis, and CTGF is responsible mainly for the maintenance of fibrotic lesions [9,10]. Because TGF-β induces the production of CTGF in a variety of cell types, we investigated the possible synergistic or additive effects of TGF-β on hypoxia-induced CTGF expression in dermal fibroblasts. As shown in Fig. 4, TGF-β increased CTGF mRNA expression dose-dependently in dermal fibroblasts. The combined treatment of suboptimal concentrations of CoCl2 (15 µM and 30 µM) and TGF-β (0·1 ng/ml and 0·5 ng/ml) additively increased the expression of CTGF mRNA compared with either CoCl2 or TGF-β alone.

Fig. 4
Transforming growth factor (TGF)-β additively increases hypoxia-induced connective tissue growth factor (CTGF) mRNA expression in dermal fibroblasts. Normal dermal fibroblasts were cultured with suboptimal concentrations of CoCl2 (15 µM ...

Effect of interferon (IFN)-γ on the CoCl2- or TGF-β-induced CTGF mRNA expression

IFN-γ has been used for the treatment of skin fibrosis in some patients with SSc [38,39]. In addition, IFN-γ inhibits collagen synthesis in vitro cultures of normal and scleroderma fibroblasts [40,41]. Thus, we tested whether IFN-γ would modulate CTGF mRNA expression in skin fibroblasts stimulated with hypoxia or TGF-β. The results showed that IFN-γ dose-dependently inhibited TGF-β-induced CTGF mRNA expression to basal levels, while it failed to down-regulate CoCl2-induced CTGF mRNA expression (Fig. 5), suggesting that IFN-γ may selectively block the pathway induced by TGF-β stimulation without affecting the signals associated with hypoxia.

Fig. 5
Interferon (IFN)-γ down-regulates connective tissue growth factor (CTGF) mRNA expression induced by transforming growth factor (TGF)-β, but not by hypoxic stimulation. Normal dermal fibroblasts were incubated with CoCl2 (100 µM) ...

CoCl2-induced CTGF mRNA expression is dependent on NF-κB activity

To determine the signalling pathway for CTGF mRNA induced by hypoxia, we investigated the effects of several signal inhibitors on CoCl2-induced CTGF mRNA expression. As shown in Fig. 6a, the SP-1 inhibitor mithramycin and the AP-1 inhibitor curcumin (10 µM) did not alter the CoCl2-induced CTGF mRNA in dermal fibroblasts, and was only partially blocked by the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB-203580. However, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) (300 µM), almost completely abrogated CTGF mRNA stimulation with CoCl2, suggesting that NF-κB is a major pathway responsible for hypoxia-induced CTGF mRNA expression. To confirm this, we performed an EMSA for the NF-κB recognition sequence in the CTGF promoter in dermal fibroblasts. As shown in Fig. 6b, CoCl2 promoted NF-κB activity in a time-dependent manner. In parallel, IκBα expression levels in the cytosol were decreased by CoCl2 treatment, suggesting that CoCl2-induced increase in NF-κB binding is mediated by suppressing IκBα expression. Together, these data indicate that NF-κB is critical for the up-regulation of CTGF mRNA expression by hypoxia.

Fig. 6
Nuclear factor (NF)-κB is the major pathway that mediates hypoxia-induced connective tissue growth factor (CTGF) mRNA expression. (a) Effect of intracellular signal inhibitors on CoCl2-induced CTGF mRNA expression. Normal dermal fibroblasts were ...

Discussion

We demonstrate here that hypoxic stimuli, such as 1% O2 and CoCl2, strongly up-regulated CTGF mRNA expression in skin fibroblasts of both SSc patients and normal controls. Moreover, transfection of the HIF-1α gene into skin fibroblasts increased CTGF expression, suggesting that chronic exposure of fibroblasts to hypoxia in SSc may elicit CTGF overproduction through HIF-1α activation. In this respect, CTGF may provide a missing link between hypoxia and excessive fibrosis in SSc. Several studies demonstrating the elevated expression of CTGF, as well as HIF-1α, in fibrotic diseases, including hepatic fibrosis, renal fibrosis and keloid, support this notion [1417].

In our previous study, serum vascular endothelial growth factor (VEGF) levels were elevated in scleroderma patients, and correlated inversely with microvascular density, as determined by nailfold capillary microscope [42]. VEGF is known to play a role in the formation of leaky and unhealthy vessels in pathological states, and acts as mitogens for fibroblasts [43]. Recently, Distler et al. have suggested that chronic and uncontrolled VEGF up-regulation is the cause of the disturbed vessel morphology in the skin of SSc patients [44]. Therefore, it can be postulated that the overproduced VEGF, triggered by hypoxia secondary to microvascular loss, may aggravate vascular injury and tissue hypoxia, which in turn induces more HIF-1α and CTGF. Alternatively, the increased activity of pErk and pAkt in SSc fibroblasts [45,46], which was caused by the chronic exposure to VEGF and/or hypoxia, may lead to the overexpression of HIF-1α and CTGF [47,48].

In fibroblasts, TGF-β stimulates collagen synthesis and myofibroblast transdifferentiation [49], and also induces the CTGF expression [50]. In the present study, exogenous TGF-β additively increased hypoxia-induced CTGF expression (Fig. 4). Considering that hypoxia stimulates TGF-β production [51], CoCl2-induced CTGF mRNA expression may have been caused by the cumulative action of CoCl2 on at least two different pathways, e.g. the direct activation of intracellular signalling, such as HIF-α induced by hypoxia, and the associated increases in TGF-β. To test the second possibility, we performed a blocking experiment using anti-TGF-β monoclonal antibodies. In reverse transcription PCR analysis, CoCl2 (100 µM) time-dependently increased TGF-β mRNA and CTGF mRNA in dermal fibroblasts until 4 h of incubation. The induction of CTGF mRNA was not blocked by simultaneous treatment with 5 µg/ml of anti-TGF-β antibodies (data not shown). In addition, cycloheximide (10 µg/ml) pretreatment failed to block the up-regulation of CTGF mRNA by CoCl2 (data not shown), suggesting that hypoxia may directly enhance CTGF transcription independent of the synthesis of TGF-β or other proteins triggered by hypoxia.

TGF-β induces CTGF expression in dermal fibroblasts via consensus Smad and transcription enhancer factor elements in the CTGF promoter [52,53]. However, HIF-1α-mediated hypoxic stimulation of CTGF expression occurs differently from TGF-β signalling [33]. In this study, CTGF expression induced by TGF-β, but not by CoCl2, was decreased by treatment with IFN-γ, suggesting that TGF-β and CoCl2 adopted different signalling pathways for CTGF induction [51]. The mechanism by which IFN-γ inhibits the TGF-β-stimulated CTGF expression is unclear. Long et al. have demonstrated that specific inhibition of STAT-3 activate Smad transcriptional responses [54], indicating that activation of STAT-3 by IFN-γ would lead to an inhibition of TGF-β/Smad-mediated CTGF transcription. Our data may explain why IFN-γ is only partially effective in relenting skin hardening in SSc patients [38,39]. Future work with other transcriptional targets regulated by IFN-γ, such as Stat 1 and 3, will help us to understand more clearly the fibrogenic mechanism of scleroderma.

Our study also demonstrated that the NF-κB inhibitor PDTC, but not an AP-1 inhibitor or p38 MAPK inhibitor, completely blocked CTGF up-regulation by CoCl2, while the NF-κB activity in the CTGF promoter is strongly up-regulated by CoCl2, indicating that NF-κB is the major pathway for CTGF induction by hypoxia. Considering that HIF-1α is critical for the activation of NF-κB [55], hypoxic stimuli may up-regulate NF-κ-mediated CTGF expression through the activation of HIF-1α without being affected by TGF-β signalling. However, current evidence suggests that the NF-κB pathway is activated in response to hypoxia independently of HIF-1α through tyrosine phosphorylation of IκBα[56]. Hypoxia-induced CTGF up-regulation could therefore be through an HIF-1α-independent mechanism. It would be interesting to test whether hypoxic stimulation induces CTGF expression in scleroderma fibroblasts through NF-κB, independently of HIF-1α. We are currently investigating such as possibility.

Taken together, our study demonstrates that upon exposed to hypoxia, dermal fibroblasts increased expression of CTGF mRNA through the activation of HIF-1α. Hypoxia-induced CTGF expression was TGF-β-independent. There was an additive effect of TGF-β on CTGF expression induced by hypoxia. IFN-γ inhibited the CTGF expression induced by TGF-β, but they had little effect on hypoxia-induced CTGF expression. NF-κB was the major pathway for mediating hypoxia-induced CTGF expression. These data suggest that the chronic hypoxic state caused by microvascular alterations may contribute to the progression of fibrosis by up-regulating CTGF expression in SSc patients.

Acknowledgments

This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (0405-DB01-0104–0006).

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