Yohei Ichimura, Yoshihide Asano, Kaname Akamata, Naohiko Aozasa, Shinji Noda, Takashi Taniguchi, Takehiro Takahashi, Tetsuo Toyama, Hayakazu Sumida, Yoshihiro Kuwano, Koichi Yanaba, Yayoi Tada, Makoto Sugaya, Shinichi Sato and Takafumi Kadono

Department of Dermatology, University of Tokyo Graduate School of Medicine, Tokyo, Japan

Angiopoietin-like protein 3 (ANGPTL3), which is part of a family of secreted glycoproteins that are structurally similar to angiopoietins, is principally expressed in the liver and is involved in lipid metabolism and angiogenesis. The aim of this study was to determine the clinical significance of serum ANGPTL3 levels, measured with a specific enzyme-linked immunosorbent assay, in patients with systemic sclerosis. Serum ANGPTL3 levels correlated positively with skin score in diffuse cutaneous systemic sclerosis with a disease duration ≤ 6 years. Furthermore, the prevalence of digital ulcers was significantly higher in patients with elevated serum ANGPTL3 levels than in other patients. Moreover, among patients excluding diffuse cutaneous systemic sclerosis with disease duration ≤ 6 years, serum ANGPTL3 levels correlated positively with estimated right ventricular systolic pressure. In conclusion, ANGPTL3 may contribute to the development of progressive skin sclerosis and proliferative obliterative vasculopathy, such as digital ulcers and pulmonary vascular involvement leading to pulmonary arterial hypertension, in systemic sclerosis. Key words: systemic sclerosis; angiopoietin-like protein 3; angiogenesis; skin sclerosis; digital ulcers; pulmonary arterial hypertension.

Accepted Apr 25, 2013; Epub ahead of print Aug 27, 2013

Acta Derm Venereol 2013; 93: XX–ZZ

Yoshihide Asano or Takafumi Kadono, Department of Dermatology, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. E-mail: yasano-tky@umin.ac.jp, kadono-der@h.u-tokyo.ac.jp

Systemic sclerosis (SSc) is a multisystem autoimmune disease characterized by initial vascular injuries and resultant fibrosis of the skin and certain internal organs. Although fibroblast activation is the final consequence of SSc, autoimmune attacks and vascular injuries prior to the onset of the fibrotic response appear to play central roles in the pathogenesis of this complicated disorder (1).

A family of secreted glycoproteins structurally similar to angiopoietins was identified and designated as angiopoietin-like proteins (ANGPTLs), which consist of an N-terminal coiled-coil domain and C-terminal fibrinogen-like domain. Despite the structural similarity to angiopoietins, ANGPTLs do not bind to endothelial tyrosine kinase receptors Tie1 and Tie2, suggesting that these molecules are functionally different from angio­poietins (2). Angiopoietin-like protein 3 (ANGPTL3), a member of the ANGPTLs, is expressed principally in the liver during development and in adults (3). ANGPTL3 is involved in lipid metabolism and angiogenesis in a domain-dependent manner. The N-terminal coiled-coin domain regulates serum lipid levels by inhibiting lipolysis of triglyceride-rich lipoproteins in mice (4, 5), while the C-terminal fibrinogen-like domain induces endothelial cell adhesion and migration and in vivo angiogenesis by interacting with endothelial integrin αVβ3 (6). In humans, serum ANGPTL3 levels correlate positively with the intima-media thickness of carotid and femoral arteries in healthy subjects, but not with triglyceride, low-density lipoprotein, high-density lipoprotein, and total cholesterol levels. More importantly, serum ANGPTL3 levels still show a positive association with arterial wall thickness after adjustment for other possible confounding variables, suggesting that the angiogenic property of ANGPTL3 explains its association with arterial wall thickness (7). Thus, unlike animal models, the role of ANGPTL3 in lipid metabolism is controversial in humans, but it definitely contributes to the development of atherosclerosis, probably by activating angiogenesis.

Given that aberrant activation of angiogenesis underlies the pathogenesis of vasculopathy and subsequent fibroblast activation in SSc, ANGPTL3 may be involved in the mechanism responsible for the pathological process in this disorder. In an initial study of this issue, we investigated serum ANGPTL3 levels and their association with clinical features in SSc.

MATERIALS AND METHODS

Patients

Serum samples, frozen at –80ºC until assayed, were obtained from 51 patients with SSc (48 women, 3 men; age, median [25–75 percentile]: 59 years [51.5–66.5]; disease duration, 3 years [2–10]) and 18 healthy individuals (16 women, 2 men; age, 54.5 years [48.3–61.8]), who visited our department between July 2009 and June 2011. Patients treated with corticosteroids or other immunosuppressants prior to their first visit were excluded. There were no significant differences in age and disease duration between men and women (age 59 [26–66] vs. 59 years [51.3–67], p = 0.66, Mann–Whitney U test; disease duration, 1 year [1–2] vs. 3.5 years [2–10], p = 0.066, Mann–Whitney U test). Patients were grouped by the LeRoy’s classification system (8): 24 patients with limited cutaneous SSc (lcSSc) (all women; age, 65 years [56.5–70.3]; disease duration, 8 years [2.8–15]) and 27 with diffuse cutaneous SSc (dcSSc) (24 women, 3 men; age 57 years [46–61]; disease duration, 2 years [2–4]). dcSSc patients were significantly younger than lcSSc patients (p = 0.0073, Mann–Whitney U test) and disease duration was much shorter in dcSSc patients than in lcSSc patients (p = 0.036, Mann–Whitney U test). All dcSSc and 22 lcSSc patients fulfilled the criteria proposed by the American College of Rheumatology (9). Two lcSSc patients not meeting these criteria had sclerodactyly and at least 2 other features of SSc, such as calcinosis, Raynaud’s phenomenon, oesophageal dysfunction, and telangiectasia. The study was performed according to the Declaration of Helsinki and approved by the ethics committee of the University of Tokyo Graduate School of Medicine. Written informed consent was obtained from all patients and healthy controls.

Measurement of serum ANGPTL3 levels

Specific enzyme-linked immunosorbent assay kits were used to measure serum ANGPTL3 levels (R&D Systems, Minneapolis, USA). Briefly, polystyrene 96-well plates coated with antibodies against ANGPTL3 were incubated with 100 µl 50-fold diluted serum at room temperature for 2 h. The wells were then washed and incubated at room temperature for 2 h with horseradish peroxidase conjugated antibodies against ANGPTL3. Next, the wells were washed again, added with tetramethylbenzidine, and incubated at room temperature for 30 min. Finally, sulphuric acid was added to terminate the reaction and the absorbance at 450 nm was measured. Serum ANGPTL3 levels were calculated using standard curve.

Clinical assessment

Disease onset was defined as the first clinical event of SSc other than Raynaud’s phenomenon. Disease duration was defined as the interval between onset and time of blood sampling. The clinical and laboratory data were obtained when the blood samples were drawn. Skin score was measured using modified Rodnan total skin thickness score (MRSS) (10). The degree of interstitial lung disease (ILD) was evaluated by the percentage of predicted vital capacity (%VC) and the percentage of predicted diffusion lung capacity for carbon monoxide (%DLco) on pulmonary function test. Ground-glass opacity on chest computed tomography was scored by 2 independent readers, as previously reported (11) and the mean estimate of the 2 readers was used as ground glass score. Elevated right ventricular systolic pressure (RVSP) was defined as 35 mmHg or more on echocardiogram. Scleroderma renal crisis (SRC) was defined as malignant hypertension and/or rapidly progressive renal failure.

Statistical analysis

The statistical analysis used for each experiment is described in the figure legends and Results section. Statistical significance was defined as p < 0.05.

RESULTS

Serum ANGPTL3 levels in patients with systemic sclerosis

No significant difference was found between serum ANGPTL3 levels in SSc patients and control subjects (median [25–75 percentiles]; 2.24 ng/ml [0.21–5.30] vs. 2.69 ng/ml [2.12–3.27], p = 0.51, Welch’s t-test, respectively). There was also no significant difference in serum ANGPTL3 levels among healthy controls, dcSSc (2.12 ng/ml [0.21–3.60]), and lcSSc patients (3.16 ng/ml [0.56–5.87]) (p = 0.42 by Kruskal-Wallis test) (Fig. 1). However, close examination of the distribution of serum ANGPTL3 levels in SSc patients, revealed 2 subgroups of SSc patients: those with quite low levels and those with highly elevated levels of serum ANGPTL3. This observation suggests that serum ANGPTL3 levels are linked to certain clinical features in patients with SSc.

Fig. 1. Serum angiopoietin-like protein 3 (ANGPTL3) levels in systemic sclerosis (SSc) and healthy controls. Serum ANGPTL3 levels were determined by a specific enzyme-linked immunoassay (ELISA). Multiple comparison was carried out with a Kruskal–Wallis test. Since serum ANGPTL3 levels were normally distributed in healthy controls (p =0.76, Shapiro–Wilk test), but not in dcSSc patients (p = 0.0001, Shapiro-Wilk test) and lcSSc patients (p =0.012, Shapiro-Wilk test), horizontal bars indicate median for each group. The dotted lines represent mean ± 2 SD of serum AGPTL3 levels in healthy controls, which were used as cut-off values in the following analyses.

Serum ANGPTL3 levels correlated positively with MRSS in dcSSc patients with disease duration ≤ 6 years

In order to investigate the clinical association of serum ANGPTL3 levels, we initially studied the correlation of serum ANGPTL3 levels with dermal and pulmonary fibrotic markers, including MRSS, %VC, and %DLco, because activation of angiogenic process is potentially linked to the initiation and progression of fibrosis in SSc. Since severe organ damage occurs within 5–6 years of disease onset in most cases of dcSSc, and progression to severe skin thickening seldom occurs afterwards (12), the correlation of serum ANGPTL3 levels with MRSS was also assessed in dcSSc patients with disease duration ≤ 6 years in addition to dcSSc and lcSSc patient groups. As shown in Fig. 2A, serum ANGPTL3 levels correlated significantly with MRSS in dcSSc patients with disease duration ≤ 6 years (r = 0.53, p = 0.022; Spearman’s rank correlation coefficient), but not in dcSSc and lcSSc patients (r = 0.39, p = 0.06, and r = 0.38, p = 0.11, respectively). Given that there was no correlation between serum ANGPTL3 levels and MRSS in dcSSc with disease duration > 6 years (r = –0.31, p = 0.56), serum ANGPTL3 levels may reflect the activation of angiogenic process during the development of skin sclerosis in dcSSc. On the other hand, regarding %VC and %DLco, no significant correlation with serum ANGPTL3 levels was detected in these 3 groups. We also evaluated the association of serum ANGPTL3 levels with ground glass score in these 3 groups because ground glass opacity reflects inflammation and vasculopathy associated with ILD better than %VC and %DLco, but did not see any significant correlation. Collectively, ANGPTL3 is potentially involved in the mechanism underlying skin sclerosis, but not ILD, in dcSSc.

Fig. 2. Clinical correlation of serum angiopoietin-like protein 3 (ANGPTL3) levels in systemic sclerosis (SSc). Serum ANGPTL3 levels correlated with modified Rodnan total skin thickness score (MRSS) in dcSSc patients with disease duration ≤ 6 years (A; r = 0.53, p = 0.022, Spearman’s rank correlation coefficient) and with right ventricular systolic pressure (RVSP) in SSc patients excluding dcSSc with disease duration ≤ 6 years (C; r = 0.39, p = 0.044, Spearman’s rank correlation coefficient). The solid line represents the regression line. The values of RVSP were significantly higher in SSc patients with normal and elevated serum ANGPTL3 levels than in those with descreased levels. (B; p = 0.036, Welch’s t-test). Since serum ANGPTL3 levels were normally distributed in SSc patients with decreased serum ANGPTL3 levels (p = 0.02, Shapiro-Wilk test), but not in patients with normal and elevated ANGPTL3 levels (p = 0.54, Shapiro-Wilk test), median was shown as horizontal bars.

Clinical features of SSc patients with elevated serum ANGPTL3 levels

We next investigated the association of serum ANGPTL3 levels with clinical features related to SSc vasculopathy. Since there was a subset of SSc patients with quite high serum ANGPTL3 levels, we first set the cut-off value at 4.86 ng/ml (mean +2 SD) of healthy controls) and classified SSc patients into 2 groups; patients with elevated serum ANGPTL3 levels (31% of SSc patients; 6 of 27 dcSSc patients and 10 of 24 lcSSc patients) and the other patients. Patient information and the prevalence of clinical features associated with SSc vasculopathy in these 2 groups are shown in the left-hand columns of Table I. There was no significant difference between these 2 groups in terms of sex, age, disease duration, and the frequency of dcSSc. Regarding the frequency of cutaneous vascular manifestations, including Raynaud’s phenomenon, nail-fold bleeding, telangiectasia, and pitting scars, there was no significant difference between these 2 groups. As for organ involvements associated with proliferative obliterative vasculopathy, the frequency of digital ulcers was significantly greater in patients with increased serum ANGPTL3 levels than in the other patients (40 % vs. 11%, p = 0.048), while the frequencies of SRC and elevated RVSP were comparable between these 2 groups. Thus, the increase in serum ANGPTL3 levels is associated with the development of digital ulcers, which are attributable to intimal proliferation and luminal narrowing or occlusion of small digital arteries and larger arteries (the palmar arch and radial and ulnar arteries) (13), in SSc patients.