15 research outputs found
Bosentan reverses the pro-fibrotic phenotype of systemic sclerosis dermal fibroblasts via increasing DNA binding ability of transcription factor Fli1
A Possible Contribution of Altered Cathepsin B Expression to the Development of Skin Sclerosis and Vasculopathy in Systemic Sclerosis
Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1+/− and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1+/− mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc
Serum Levels of Galectin-3: Possible Association with Fibrosis, Aberrant Angiogenesis, and Immune Activation in Patients with Systemic Sclerosis
Serum pro-CTSB levels in dcSSc patients further classified into subgroups based on disease duration.
<p>dcSSc patients were divided into 3 subgroups: those with disease duration of <1 years, those with disease duration of 1 to 6 years, and those with disease duration of >6 years. Serum pro-CTSB levels were determined by a specific ELISA. The horizontal bars indicate the mean value in each group. Statistical analysis was carried out with a Kruskal-Wallis test and a Steel-Dwass' test for multiple comparison. *P<0.05.</p
CTSB expression was up-regulated in dermal vasculature of Fli1<sup>+/−</sup> mice and in Fli1 siRNA-treated HDMECs.
<p>Immunodetection of CTSB proteins in the skin sections of 3 month-old wild type (A) and Fli1<sup>+/−</sup> (B) mice (original magnification was ×40) by Vectastain ABC kit according to the manufacturer's instruction. Insets (original magnification was ×40) depict representative arterioles (panel 1), venules (panel 2), and capillaries (panel 3; red arrowheads), respectively. Representative results in 5 wild type and 5 Fli1<sup>+/−</sup> mice are shown. (C) HDMECs were seeded shortly before transfection. The cells were transfected with 10 nM of Fli1 and scrambled non-silencing siRNA (Santa Cruz) using HiPerfect transfection reagent (Qiagen, Valencia, CA, USA) for 72 hours. Cells were then serum starved for the last 24 hours. mRNA levels of Fli1 and CTSB genes were examined by quantitative real-time PCR and normalized to the levels of human 18S rRNA gene. Results of controls or relative value compared with the controls are expressed as means ± SD of 3 independent experiments. Statistical analysis was carried out with a 2-tailed paired t-test. *P<0.05, **P<0.005.</p
CTSB expression was up-regulated in dermal vasculatures of SSc patients compared to those in controls.
<p>CTSB expression levels in dermal vasculatures were determined by immunohistochemistry in skin section from 8 healthy control subjects (A, B) and 8 SSc patients (C, D). Representative results are shown. Original magnification was ×200 (A, C) and ×400 (B, D). Analysis of CTSB expression levels in vessel walls is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032272#pone-0032272-t002" target="_blank">Table 2</a>.</p
Cathepsin B levels in dermal vasculature in normal and systemic sclerosis skin.
<p>NS, normal skin; SSc, systemic sclerosis; dcSSc, diffuse cutaneous systemic sclerosis; lcSSc, limited cutaneous systemic sclerosis. We used the following grading system: −, no staining; +, slight staining; ++, moderate staining; +++, strong staining.</p
Serum pro-CTSB levels in patients with dcSSc, lcSSc, and healthy individuals.
<p>Serum pro-CTSB levels were determined by a specific ELISA. Bars indicate the mean value in each group. Statistical analysis was carried out with a Kruskal-Wallis test and a Steel-Dwass' test for multiple comparison. *P<0.05.</p