CTSB expression was up-regulated in dermal vasculature of Fli1^+/− 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^+/− (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^+/− 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

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

Correlation of serum pro-cathepsin B levels with clinical features in patients with dcSSc and lcSSc.

<p>Unless noted otherwise, values are percentages. dcSSc, diffuse cutaneous systemic sclerosis; lcSSc, limited cutaneous systemic sclerosis; MRSS, modified Rodnan total skin thickness score; DLco, diffuse capacity for carbon monoxide; VC, vital capacity; RVSP; right ventricular systolic pressure. Patients were evaluated for the presence of esophageal, pulmonary, cardiac, renal, or muscle involvements, as follows. Esophagus hypomotility was defined as distal esophageal hypomotility on barium-contrast radiography. Interstitial lung disease (ILD) was defined as bibasilar interstitial fibrosis on chest radiographs, and in patients with no abnormalities on chest radiographs early ILD was defined as alveolitis on high-resolution computer tomography. Elevated right ventricular systolic pressure (RVSP) was defined as 35 mmHg or more on echocardiogram. Cardiac involvement was defined as any of the following: symptomatic pericarditis, clinical evidence of left ventricular congestive heart failure, or arrhythmias requiring treatment. Scleroderma renal crisis was defined as malignant hypertension and/or rapidly progressive renal failure. Skeletal muscle involvement was defined as proximal muscle weakness and elevated serum creatine kinase levels, plus abnormal electromyographic findings consistent with myopathy and/or histopathologic changes in inflammatory myopathy. Disease onset was defined as the first clinical event of SSc other than Raynaud's phenomenon. Disease duration was defined as the interval between the onset and the time the blood samples were drawn. Statistical analysis was carried out with Fisher's exact probability test.</p><p>*P<0.05.</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