A possible contribution of lipocalin-2 to the development of dermal fibrosis, pulmonary vascular involvement and renal dysfunction in systemic sclerosis.

BACKGROUND: Lipocalin-2 is an adipocytokine implicated in apoptosis, innate immunity, angiogenesis, and the development of chronic kidney disease. OBJECTIVES: To investigate the role of lipocalin-2 in systemic sclerosis (SSc). MATERIALS AND METHODS: Serum lipocalin-2 levels were determined by enzyme-linked immunosorbent assay in 50 patients with SSc and 19 healthy subjects. Lipocalin-2 expression was evaluated in the skin of patients with SSc and bleomycin (BLM)-treated mice and in Fli1-deficient endothelial cells by reverse transcriptase-real time polymerase chain reaction, immunoblotting and/or immunohistochemistry. RESULTS: Although serum lipocalin-2 levels were comparable between patients with SSc and healthy controls, the prevalence of scleroderma renal crisis was significantly higher in patients with SSc with elevated serum lipocalin-2 levels than in those with normal levels. Furthermore, serum lipocalin-2 levels inversely correlated with estimated glomerular filtration rate in patients with SSc with renal dysfunction. Among patients with SSc with normal renal function, serum lipocalin-2 levels positively correlated with skin score in patients with diffuse cutaneous SSc with disease duration of < 3 years and inversely correlated with estimated right ventricular systolic pressure in total patients with SSc. Importantly, in SSc lesional skin, lipocalin-2 expression was increased in dermal fibroblasts and endothelial cells. In BLM-treated mice, lipocalin-2 was highly expressed in dermal fibroblasts, but not in endothelial cells. On the other hand, the deficiency of transcription factor Fli1, which is implicated in SSc vasculopathy, induced lipocalin-2 expression in cultivated endothelial cells. CONCLUSIONS: Lipocalin-2 may be involved in renal dysfunction and dermal fibrosis of SSc. Dysregulated matrix metalloproteinase-9/lipocalin-2-dependent angiogenesis due to Fli1 deficiency may contribute to the development of pulmonary arterial hypertension associated with SSc.

Histological features of localized scleroderma 'en coup de sabre': a study of 16 cases.

BACKGROUND: Early lesions of localized scleroderma are histologically characterized by perivascular lymphocytic infiltrate in the reticular dermis and swollen endothelial cells. However, there have been few information regarding histological features other than these findings in localized scleroderma. OBJECTIVE: Since en coup de sabre (ECDS) is a certain subset of localized scleroderma with a relatively uniform clinical manifestation, we focused on this disease subset and evaluated its histopathological features. METHODS: A total of 16 patients with ECDS were retrospectively evaluated on the basis of clinical and histological findings. RESULTS: Regardless of clinical manifestations, vacuolar degeneration was found in all of the ECDS patients. Importantly, keratinocyte necroses were restricted to early and active ECDS lesions. In early ECDS patients (disease duration of <3 years), moderate to severe perivascular and/or periappendageal lymphocytic infiltrate and vacuolar changes in follicular epithelium were more prominent, whereas epidermal atrophy was less frequently observed, than in late ECDS patients (disease duration of ≥6 years). CONCLUSION: Vacuolar degeneration at the dermoepidermal junction is a common histological feature in ECDS and perivascular and/or periappendageal lymphocytic infiltrate and vacuolar degeneration of follicular epithelium are characteristic especially in early ECDS, further supporting a canonical idea that the elimination of mutated epidermal cells by immune surveillance contributes to tissue damage and resultant fibrosis in localized scleroderma.

Serum levels of ADAM12-S: possible association with the initiation and progression of dermal fibrosis and interstitial lung disease in patients with systemic sclerosis.

BACKGROUND: A disintegrin and metalloprotease (ADAM) 12 is one of the metalloproteinase-type ADAMs and possesses extracellular metalloprotease and cell-binding functions. ADAM12 is expressed in two alternative forms, such as a membrane-anchored form (ADAM12-L) and a short secreted form (ADAM12-S). OBJECTIVE: To investigate the clinical significance of serum ADAM12-S levels in systemic sclerosis (SSc). METHODS: Serum ADAM12-S levels were determined by a specific enzyme-linked immunosorbent assay in 61 SSc patients and 18 healthy controls. RESULTS: Serum ADAM12-S levels were significantly increased in diffuse cutaneous SSc (dcSSc) patients than in healthy controls (0.417 ± 0.389 vs. 0.226 ± 0.065 ng/mL; P < 0.05), while being comparable between limited cutaneous SSc (0.282 ± 0.258 ng/mL) and healthy controls. Serum ADAM12-S levels significantly elevated in dcSSc patients with disease duration of ≤ 6 years (0.537 ± 0.449 ng/mL, P < 0.05), but not in dcSSc with disease duration of >6 years (0.225 ± 0.049 ng/mL), compared to healthy controls. Furthermore, in dcSSc patients with disease duration of ≤ 6 years, serum ADAM12-S levels correlated positively with modified Rodnan total skin thickness score, ground glass score, and serum C-reactive protein values, while showed inverse correlation with fibrosis score. CONCLUSION: Elevated serum ADAM12-S levels are associated with elevated serum inflammatory marker, severity of skin fibrosis, and activity of interstitial lung disease in dcSSc, suggesting the possible contribution of ADAM12-S to the pathological events in this disorder.

Clinical significance of serum retinol binding protein-4 levels in patients with systemic sclerosis.

BACKGROUND: Retinol binding protein-4 (RBP-4) is a member of adipocytokines, which is potentially associated with fibrosis, vasodilation, and angiogenesis in addition to insulin resistance. OBJECTIVE: To investigate the clinical significance of serum RBP4 levels in patients with systemic sclerosis (SSc), which is a systemic autoimmune disease characterized by fibrosis and vasculopathy. METHODS: Serum RBP4 levels were determined by enzyme-linked immunosorbent assay in 62 SSc patients and 19 healthy controls. RESULTS: Similar to patients with chronic kidney disease, serum RBP4 levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction. Therefore, analyses were carried out by excluding SSc patients with estimated glomerular filtration rate <60 mL/min/1.73 m(2) . Serum RBP4 levels were significantly lower in diffuse cutaneous SSc (dcSSc) than in control subjects [median (25-75 percentile); 25.8 µg/mL (19.6-47.0) vs. 43.1 µg/mL (31.7-53.4), P < 0.05], while there was no significant difference between limited cutaneous SSc (lcSSc) [28.0 µg/mL (25.4-43.3)] and control subjects. In both of dcSSc and lcSSc, patients with Raynaud's phenomenon had RBP4 levels significantly lower than those without. Furthermore, serum RBP4 levels inversely correlated with pulmonary function test results in dcSSc and with right ventricular systolic pressure in lcSSc. CONCLUSION Decreased RBP4 levels are associated with the prevalence of Raynaud's phenomenon in dcSSc and lcSSc, with the severity of interstitial lung disease in dcSSc, and with the degree of pulmonary vascular involvement in lcSSc, suggesting the possible contribution of RBP4 to the pathological events in this disorder.

Serum apelin levels: clinical association with vascular involvements in patients with systemic sclerosis.

BACKGROUND: Apelin is a bioactive peptide exerting its pro-angiogenic and pro-fibrotic effects in a context-dependent manner through the activation of its receptor APJ, which is ubiquitously expressed on the surface of various cell types. The activation of apelin/APJ signalling appears to be involved in the pathological process of fibrotic disorders, including liver cirrhosis. OBJECTIVE: As an initial step to clarify the role of apelin/APJ signalling in the pathogenesis of systemic sclerosis (SSc), we investigated serum apelin levels and their clinical association in patients with SSc. METHODS: Serum apelin levels were determined by a specific enzyme-linked immunosorbent assay in 56 SSc patients and 18 healthy controls. RESULTS: Serum apelin levels were comparable among three groups, including diffuse cutaneous SSc, limited cutaneous SSc and control subjects (1.77 ± 1.48, 1.63 ± 1.51 and 1.61 ± 0.44 ng/mL, respectively). When we classified SSc patients into three groups according to disease duration, serum apelin levels were elevated in early SSc (<3 years) compared with mid-stage SSc (3-10 years) (1.74 ± 1.26 vs. 1.02 ± 0.52 ng/mL, P < 0.05). Importantly, in late stage SSc (>10 years), the prevalence of severe vascular involvements, including intractable skin ulcers, scleroderma renal crisis and pulmonary arterial hypertension, was significantly higher in patients with elevated serum apelin levels than in those without (100% vs. 20%, P < 0.05). CONCLUSION: Apelin may be associated with altered and activated angiogenesis prior to fibrotic responses in early SSc and with the development of proliferative vasculopathy in late stage SSc.

Constitutive activation of c-Abl/protein kinase C-δ/Fli1 pathway in dermal fibroblasts derived from patients with localized scleroderma.

BACKGROUND: A noncanonical pathway of transforming growth factor-ß signalling, the c-Abl/protein kinase C-δ (PKC-δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts. OBJECTIVES: To investigate the significance of the c-Abl/PKC-δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc). METHODS: The activation status of the c-Abl/PKC-δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet-derived growth factor receptor inhibitor AG1296 and gene silencing of c-Abl on the expression levels of type I collagen were evaluated by immunoblotting. RESULTS: The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c-Abl were elevated compared with cultured normal fibroblasts and PKC-δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c-Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts. CONCLUSIONS: Constitutive activation of the c-Abl/PKC-δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc.

Augmented production of soluble CD93 in patients with systemic sclerosis and clinical association with severity of skin sclerosis.

BACKGROUND: The cell surface protein CD93, expressed on endothelial and myeloid cells, mediates phagocytosis, inflammation and cell adhesion. A soluble form of CD93 (sCD93) is released during inflammation. OBJECTIVES: To determine the serum sCD93 level and its association with clinical parameters in patients with systemic sclerosis (SSc). METHODS: Serum sCD93 levels were examined by enzyme-linked immunosorbent assay in 59 patients with SSc, 24 patients with systemic lupus erythematosus and 47 healthy individuals. The expression of CD93 in skin tissues was examined immunohistochemically. In a retrospective longitudinal study, sera from 11 patients with SSc were analysed. RESULTS: Serum sCD93 levels were increased in patients with SSc compared with healthy individuals (P<0·001). Patients with diffuse cutaneous SSc showed greater levels of sCD93 than those with limited cutaneous SSc (P<0·01) or systemic lupus erythematosus (P<0·01). Serum sCD93 levels correlated positively with the severity of skin sclerosis. Strong CD93 immunostaining was observed on endothelial cells in lesional skin tissues. In the longitudinal study, sCD93 levels decreased in parallel with improvement in skin sclerosis. CONCLUSIONS: Serum sCD93 levels are increased in patients with SSc and correlate with the severity and activity of skin sclerosis. CD93 may contribute to the development of skin fibrosis in SSc.

Serum adiponectin levels inversely correlate with the activity of progressive skin sclerosis in patients with diffuse cutaneous systemic sclerosis.

BACKGROUNDS: Adiponectin has been demonstrated to be one of anti-inflammatory and anti-fibrotic factors, suggesting the potential of this cytokine to be involved in the developmental process of systemic sclerosis (SSc). OBJECTIVE: The aim of this study was to investigate the clinical significance of serum adiponectin levels in patients with SSc. Methods Serum adiponectin levels were determined using enzyme-linked immunosorbent assay (ELISA) in 32 patients with diffuse cutaneous SSc (dcSSc), 28 with limited cutaneous SSc (lcSSc) and 27 healthy controls. No significant difference between these groups existed in terms of gender, age and body mass index. RESULTS: Serum adiponectin levels in dcSSc patients (4.93 ± 6.48 µg/mL) were significantly lower than those in lcSSc patients (9.69 ± 7.61 µg/mL, P < 0.01) and healthy controls (9.36 ± 5.57 µg/mL, P < 0.01). dcSSc patients with disease duration of ≤5 years had significantly decreased serum adiponectin levels (2.15 ± 1.69 µg/mL) than those with disease duration of >5 years (13.29 ± 8.36 µg/mL, P < 0.01), lcSSc patients with disease duration of ≤5 years (8.07 ± 7.98 µg/mL, P < 0.05), lcSSc patients with disease duration of >5 years (10.9 ± 7.34 µg/mL, P < 0.01) and healthy controls (9.36 ± 5.57 µg/mL, P < 0.01). Longitudinal studies in five patients with early dcSSc treated with oral prednisone demonstrated that serum adiponectin levels inversely correlate with the activity of progressive skin sclerosis in dcSSc patients. CONCLUSION: Serum levels of adiponectin may serve as a useful marker to evaluate the activity of progressive skin sclerosis in dcSSc.

Serum Tie2 levels: clinical association with microangiopathies in patients with systemic sclerosis.

BACKGROUND: Tie2 and its ligand, angiopoietins (Ang), regulate the transition between vascular quiescence and angiogenesis. Although defective angiogenesis is one of the major causes of microangiopathies in systemic sclerosis (SSc), the role of Ang/Tie2 signalling in the development of SSc has never been examined. OBJECTIVE: To investigate the clinical significance of the soluble Tie2 domain (sTie2) in serum samples from SSc patients. METHODS: Serum sTie2 levels were determined by a specific enzyme-linked immunosorbent assay in 48 SSc patients and nine normal controls. RESULTS: There was no significant difference in serum sTie2 levels between SSc patients and healthy controls (14.8 ± 3.4 vs. 14.7 ± 1.1 ng/mL). When we set the cut-off value at 16.97 ng/mL (mean + 2SD) based on the data of normal controls, 27% of SSc patients showed elevated serum sTie2 levels. The frequencies of nailfold bleeding and pulmonary arterial hypertension (PAH) were significantly higher in patients with increased serum sTie2 levels than in those with sTie2 levels not elevated (70% vs. 47% and 60% vs. 21%, respectively, P < 0.05). There was also a trend towards the elevation of serum sTie2 levels in SSc patients with PAH compared to those without; however, it did not reach statistical significance (16.7 ± 3.6 vs. 14.2 ± 3.4 ng/mL, P = 0.059). CONCLUSION: Soluble Tie2 domain (sTie2) may be related to the development of vascular abnormalities in SSc, possibly by modulating the Ang/Tie2-mediated angiogenic process. Furthermore, the serum sTie2 levels may serve as a useful marker for SSc-related PAH, contributing to early diagnosis and therapeutic intervention.

Identification and characterization of a Drosophila homologue of ATBP.

ATBP [(A+T)-stretch binding protein] activates the Sarcophaga defense protein genes in an (A+T)-stretch dependent manner as shown previously using a luciferase reporter assay [Aozasa et al. (2001) Eur J Biochem, 268, 2506-2511]. We identified the Drosophila homologue of ATBP. Drosophila ATBP has 388 amino acid residues and the amino acid sequence has high similarity with that of the Sarcophaga ATBP. In particular, residues 1-56 and 301-374 are highly conserved between Sarcophaga and Drosophila. Drosophila ATBP activated the Sarcophaga lectin gene promoter in SL-2 cells. The Drosophila ATBP gene is a single copy gene and is located in the 20E region of chromosome X.

Interaction between ATBP and DmUbc9 in the expression of the Sarcophaga lectin gene.

We previously demonstrated that (A+T)-stretch binding protein (ATBP) and Dorsal-related immunity factor (Dif) are required for the expression of the Sarcophaga lectin gene in SL-2 cells (Aozasa et al., Eur. J. Biochem. 268, 2506-2511, 2001). The present study demonstrates that DmUbc9 interacts with ATBP, and cotransfection of the DmUbc9 vector with ATBP and Dif vectors greatly enhances the expression of the luciferase reporter of the Sarcophaga lectin gene in SL-2 cells. These results suggest that sumoylation of ATBP is involved in the expression of the Sarcophaga lectin gene in this system.

Activation of the Sarcophaga lectin gene promoter by (A + T)-stretch binding protein.

Previously, we purified and isolated a cDNA for (A + T)-stretch binding protein (ATBP) that binds to (A + T)-stretches in the 5' upstream region of the Sarcophaga lectin gene [Nakanishi-Matsui, M., Kubo, T. & Natori, S. (1995) Eur. J. Biochem. 230, 396-400]. Here, we used a luciferase reporter to examine the effect of ATBP on transcription of the Sarcophaga lectin gene. Deletion experiments revealed that ATBP activates the Sarcophaga lectin gene in a 5' upstream sequence-dependent manner, and that at least the N-terminal 25 residues, the three Zn-finger domains, an acidic domain and the third hydrophobic domain of ATBP are indispensable for its function. Furthermore, a synergistic effect was detected between ATBP and Dif, suggesting that ATBP is involved in the activation of insect immunity genes.

Molecular cloning and characterization of SRAM, a novel insect rel/ankyrin-family protein present in nuclei.

Previously, we purified a 59-kDa protein that binds to the kappaB motif of the Sarcophaga lectin gene. Here we report its cDNA cloning and some of its characteristics as a novel member of the Rel/Ankyrin-family. This protein, named SRAM, contained a Rel homology domain, a nuclear localization signal and 4 ankyrin repeats, but lacked the Ser-rich domain and PEST sequence that Relish contained. We found that SRAM was localized in the nuclei of NIH-Sape-4 cells, which are an embryonic cell line of Sarcophaga. The Sarcophaga lectin gene promoter containing tandem repeats of the kappaB motifs was activated in NIH-Sape-4 cells. In Drosophila mbn-2 cells, Dif alone activated this reporter gene and a cooperative effect was detected when SRAM and Dif were co-transfected, although SRAM alone did not activate it. This is the first report of a Rel/Ankyrin molecule that exists in the nuclei.