Background
Systemic sclerosis (SSc) is a multisystem connective tissue disorder that typically results in fibrosis of the skin and internal organs [
1]. SSc is characterized by autoimmunity, inflammation, and widespread microvascular injury, leading to the activation of fibroblasts and excessive accumulation of extracellular matrix (ECM) proteins; however, the causes and the pathogenesis of this disease are not yet fully explained [
2]. Accumulation of ECM proteins restricts normal function of the affected tissues and organs leading to high morbidity and mortality. Despite significant improvements in the management of SSc, effective disease modifying therapies are not yet available.
Connective tissue growth factor (CTGF, also known as CCN2), a member of the CCN family of matricellular proteins, is widely known as a hallmark of fibrosis in multiple tissues, including skin, heart, lung, liver, and kidney [
3,
4]. A key study in support of the essential role of CTGF in fibrosis was published by Takehara and colleagues who showed that subcutaneous injection of transforming growth factor (TGF)-β and CTGF led to sustained fibrosis, while either factor alone failed to do so [
5]. Further support was provided by a recent study that showed that fibroblast-specific ablation of CTGF inhibited development of dermal fibrosis in the bleomycin injection model [
6]. The role of CTGF in fibrosis may not be limited to its function as a co-factor of TGF-β, but CTGF may also regulate other aspects of the fibrogenic process, consistent with its multifunctional nature [
7,
8]. Accordingly, it has been suggested that CTGF may contribute to myofibroblast recruitment during bleomycin-induced skin fibrosis [
6]. Transgenic mice overexpressing CTGF develop skin fibrosis and microvascular abnormalities [
9], and loss of CTGF results in a reduction of bleomycin-induced skin fibrosis [
6].
Accumulating reports show that CTGF is highly expressed in SSc [
10]. Elevated levels of CTGF have been identified in fibrotic skin and serum from patients with SSc, and have been correlated with the severity of skin and lung fibrosis [
11,
12]. Elevated CTGF protein expression has been observed in fibroblasts in affected skin in SSc [
13]. Furthermore, N-terminal cleavage products of CTGF have been identified in interstitial fluid in the skin of patients with SSc [
14].
Given the key role of CTGF in the development of skin fibrosis in SSc, the goal of this study was to evaluate the efficacy of FG-3019 as a potential therapeutic agent for SSc using a murine model of angiotensin II (Ang II)-induced skin fibrosis [
15‐
17]. FG-3019 is a fully human monoclonal antibody specific for CTGF [
18]. Clinically, FG-3019 is being evaluated for treatment of idiopathic pulmonary fibrosis, pancreatic cancer, and Duchenne muscular dystrophy [
19‐
21]. In parallel studies, we employed mice with smooth muscle cell fibroblast-specific deletion of CTGF to assess the contribution of CTGF to the process of skin fibrosis in the Ang II model [
6]. Together, these studies showed that FG-3019 was comparable to genetic CTGF deletion in attenuating skin fibrosis. The results of this study support the use of FG-3019 as therapy for skin fibrosis.
Methods
Reagents
A fully human IgG1kappa monoclonal antibody recognizing domain 2 of human and rodent CTGF (FG-3019) and whole human IgG control antibody were obtained from Fibrogen (San Francisco, CA, USA).
Mice
Smooth muscle cell fibroblast-specific CTGF knockout (KO) mice were generated as described [
6]. In brief, mice with CTGF flanked by loxP sites were crossed with mice containing a Cre recombinase gene located downstream of a collagen α2 (I) promoter enhancer that confers fibroblast-specific gene expression. The expression of Cre is dependent on the presence of tamoxifen. Tamoxifen was diluted in corn oil to 10 mg/ml. Three-week-old mice were given intraperitoneal injections of the tamoxifen suspension (100 μl of 10 mg/ml) over 10 days. Three weeks later, the mice were used for further studies. We used mice homozygous for CTGF gene flanked by loxP sites and heterozygous for Cre as CTGF KO mice. Littermate mice homozygous for loxP-flanked CTGF that were wild for Cre (non Cre) were used as control mice. C57BL/6 J mice for FG-3019 injection were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All mice experiments were performed in accordance with the National Institutes of Health and institutional guidelines for animal care, and approved by the Committee on the Ethics of Animal Experiments of the Boston University (Protocol AN-15037).
Cell culture and immunoblotting
Mouse dermal fibroblasts were obtained from skin from the backs of male CTGF KO and control littermate mice. Fibroblasts between the third and fifth sub passages were used for experiments. Mouse dermal fibroblasts were lysed and subjected to immunoblotting, as described previously [
22]. Primary antibodies used were (1:1000): CTGF from Santa Cruz Biotechnology (Dallas, TX, USA) and β-actin from Sigma (St. Louis, MO, USA).
Ang II-induced dermal fibrosis
Alzet osmotic miniature pumps (Model 1002, DURECT, Cupertino, CA, USA) delivering Ang II (EMD Millipore, Billerica, MA, USA) at a rate of 1000 ng/kg/min (pressor dose) or PBS, were implanted subcutaneously on the back of 8-week-old mice, as described previously [
17]. After 2 weeks, mice were euthanized and the skin surrounding the pump outlet was collected. FG-3019 (25 mg/kg) or control IgG (25 mg/kg) was administered intraperitoneally three times per week for 2 weeks, after the osmotic pump was installed.
Histologic assessment
Mice skin samples were paraffin-embedded, and sections (5 μm in thickness) were stained with hematoxylin and eosin (HE). Dermal thickness was evaluated by measuring the distance between the epidermal-dermal junction and the dermal-fat junction in HE sections. Skin trichrome staining was performed by Masson’s trichrome stain kit (Polysciences, Warrington, PA, USA). The αSMA positive cells were counted in five random high-power fields using a light microscope. The mean score was used for analysis. The von Willebrand factor (vWF) staining intensity for immunohistochemical assessment was scored semi-quantitatively. The staining intensity (1: negative or weak staining, 2: moderate staining, and 3: strong staining) was evaluated in six randomly selected fields in the subcutaneous area. Then a semi-quantitative score per sample was generated by calculating the average of the six intensity scores per sample.
Hydroxyproline assay
Collagen deposition was determined by measuring total hydroxyproline content in 8-mm skin punch biopsies obtained from PBS and Ang II infusion sites as described previously [
16]. Briefly, the skin samples were hydrolyzed with 6 M sodium hydroxide at 120 °C for 16 h. The hydrolysate was then oxidized with oxidation buffer (one part 7% chloramine T and four parts acetate citrate buffer) for 4 minutes at room temperature. Ehrlich’s aldehyde reagent was added to each sample, and the chromophore was developed by incubating the samples at 60 °C for 25 minutes. Absorbance of each sample was read at 560 nm using a spectrophotometer. Results were expressed as relative hydroxyproline content. A standard curve was performed for all hydroxyproline measurements using known quantities of hydroxyproline.
Immunohistochemical assessment
For single antibody staining, formalin-fixed, paraffin-embedded 5-μm skin tissue sections were de-paraffinized and rehydrated through a graded series of ethanol. Antigens were retrieved by incubation with a proteinase K solution (EMD Millipore, Billerica, MA, USA) for 5 minutes. Blocking was done by 2.5% normal horse serum for 1 h. Tissue sections were then incubated with primary antibody to αSMA (1:100, Novus Biologicals, Littleton, CO, USA), PDGFRβ (1:50, Cell Signaling Technology, Danvers, MA, USA), rat anti-mouse CD45 Ab (1:100, BD Pharmingen, San Diego, CA, USA), phospho-Smad2 (Ser465/467) Ab (1:100, Cell Signaling), or vWF (1:1000, DAKO, Santa Clara, CA, USA) at 4 °C for 16 h. Next, sections were incubated with ImmPress horseradish peroxidase (HRP) anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) for 30 minutes. The color was developed using 3,3-diaminobenzidine (DAB) substrate (DAKO). Immunohistochemical images were collected using a microscope (BH-2; Olympus, Center Valley, PA, USA).
Immunofluorescence staining
Staining was performed on 5-μm paraffin sections or cryosections. Slides were blocked with a blocking solution (3% BSA, and 0.2% Triton X-100 in PBS) for 2 h. Then, tissue sections were incubated at 4 °C overnight with primary antibodies: procollagen (COL1A1 propeptide, 1:50, Thermo Fisher Scientific) for the cryosections and rabbit anti-mouse FSP1 (1:100, Abcam, Cambridge, MA, USA) for the paraffin sections. Secondary antibodies conjugated with Alexa 594 (Thermo Fisher Scientific) were used. Coverslips were mounted by using Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Fluorescence images were recorded with FV10i fluorescence microscope (Olympus, Tokyo, Japan).
Statistical analysis
Values are presented as means ± standard deviation (SD). One-way analysis of variance with Tukey-Kramer test was used to determine significant differences between more than two groups. Analyses were performed with Statcel software (OMS, Tokorozawa, Japan). Significance was defined as P <0.05.
Discussion
Currently, there are only limited treatment options for patients with SSc. Recent studies have provided evidence of an important role of CTGF in the development of organ fibrosis, including the skin [
25]. Blockade of CTGF using the human anti-CTGF antibody, FG-3019, effectively prevented the development of skin fibrosis in response to Ang II challenge. The anti-fibrotic effect of FG-3019 was comparable to the genetic deletion of CTGF in collagen-producing cells. In addition, both treatments were very effective in reducing inflammation and lessening the damaging effects of Ang II on skin microvasculature.
The current study supports the findings of Liu et al. [
6] who demonstrated the essential role of CTGF in bleomycin-induced fibrosis. In both studies, deletion of CTGF from fibroblasts and smooth muscle cells/pericytes prevented increased collagen deposition and myofibroblast accumulation in the skin. In addition, we also showed that blockade of CTGF significantly reduced the number of PDGFRβ-positive cells in the fibrotic lesion. Although the origin of these PDGFRβ collagen-producing cells in this model is not known, these cells may represent the expansion of local fibroblasts. Alternatively, they could originate from pericytes or mesenchymal stem cells. The role of CTGF in recruitment and expansion of these cells is not clear at present. Side by side comparison of the anti-fibrotic effects of FG-3019 and genetic deletion of CTGF showed that antibody directed against CTGF was comparable to CTGF deletion in blocking the CTGF profibrotic function in this study. Our results are also consistent with previous findings that showed therapeutic effects of FG-3019 in bleomycin-induced lung fibrosis [
26].
Although profibrotic functions of CTGF are most widely recognized, CTGF has also been shown to induce inflammatory response in various cell types in vitro [
7]. The pro-inflammatory role of CTGF was further confirmed in in vivo models of pancreatic and renal inflammation [
27,
28]. In our model of Ang II-induced fibrosis, which is also characterized by increased inflammation, blockade of CTGF almost completely abrogated infiltration of inflammatory cells, including fibrocytes. Interestingly, treatment with FG-3019 appeared to be more potent than genetic CTGF deletion in reducing infiltration of CD45-positive cells and pSmad2 cells. However, both treatments were similarly effective in reducing the number of fibrocytes. These findings are consistent with a recent report that showed an inhibitory effect of FG-3019 on acute and delayed immune responses in a model of radiation-induced pulmonary fibrosis [
29], where the effect appears to be indirect via modulation of myofibroblast activation and chemokine secretion (K. Lipson, personal communication). Thus, in this study, it is conceivable that blockade of CTGF inhibits recruitment of inflammatory cells from the circulation either by preventing secretion of the pro-inflammatory chemokines/cytokines by the activated resident cells or by reducing vascular injury and vessel permeability.
Ang II has a detrimental effect on the vasculature by affecting both endothelial and vascular smooth muscle cells (vSMCs) [
30]. CTGF plays an important role in the activation of vSMCs by promoting their growth, migration, and production of collagen. Furthermore, Ang II is known to induce CTGF in vSMCs [
8]. It has also been reported that CTGF is overexpressed in microvascular endothelial cells in SSc and that conditioned medium from microvascular endothelial cells stimulates the proliferation and migration of fibroblasts in SSc [
31]. Although the association between CTGF and cardiovascular diseases is well-documented [
8,
32], less is known about the potential contribution of CTGF to vascular disease in SSc. In this study, we performed a limited assessment of the effects of CTGF blockade on vascular damage by measuring the vascular injury marker, vWF. We showed that inhibition of CTGF partially reduced vWF expression. The effect was more pronounced in the CTGF KO than in FG-3019-treated mice. Together, these data suggest that blockade of CTGF may have a beneficial effect on vascular injury. The limitation of this study is that it only assessed the preventive effect of FG-3019, and further studies are needed to evaluate whether FG-3019 is effective in reversing the established fibrosis. A phase II clinical trial evaluating FG-3019 as a treatment for idiopathic pulmonary fibrosis has recently been completed. Considering the anti-fibrotic effects of FG-3019 in the skin, our study strongly supports the testing of FG-3019 as a therapeutic agent for SSc dermal fibrosis.
Acknowledgements
Not applicable.
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