A potential contribution of psoriasin to vascular and epithelial abnormalities and inflammation in systemic sclerosis.

BACKGROUND: Antimicrobial peptides have attracted much attention as a member of disease-associated molecules in systemic sclerosis (SSc), which is pathologically characterized by immune abnormalities, vasculopathy and tissue fibrosis. OBJECTIVE: To investigate the potential contribution of one of the antimicrobial peptide psoriasin to the development of SSc. METHODS: Psoriasin expression in the skin samples and sera derived from SSc patients and its correlation with clinical parameters were analysed. Psoriasin expression was evaluated by immunohistochemistry with skin samples from SSc patients and healthy controls. Serum levels of psoriasin were determined by enzyme-linked immunosorbent assay in 51 SSc patients and 19 healthy controls and assessed for the association with clinical symptoms. RESULTS: The expression of psoriasin was elevated in the epidermis of SSc lesional skin. Serum psoriasin levels were higher in SSc patients, especially in diffuse cutaneous SSc patients with disease duration of <6 years, than in healthy controls. With respect to clinical association, SSc patients with interstitial lung disease, telangiectasia and pitting scars had significantly augmented levels of serum psoriasin than those without each of these symptoms. In the subgroup of patients with interstitial lung disease, the elevation of serum psoriasin levels was associated with higher ground-glass opacity scores. Furthermore, serum psoriasin levels were decreased after the treatment with intravenous cyclophosphamide pulse as compared to baseline values. CONCLUSION: Our findings indicate a possible contribution of psoriasin to the development of clinical symptoms associated with vascular and epithelial abnormalities and inflammation in SSc, further supporting the roles of antimicrobial peptides in the SSc pathogenesis.

A potential contribution of antimicrobial peptide LL-37 to tissue fibrosis and vasculopathy in systemic sclerosis.

BACKGROUND: LL-37 is an antimicrobial peptide with pleiotropic effects on the immune system, angiogenesis and tissue remodelling. These are cardinal pathological events in systemic sclerosis (SSc). OBJECTIVES: To elucidate the potential role of LL-37 in SSc. METHODS: The expression of target molecules was evaluated by immunostaining and quantitative reverse-transcription real-time polymerase chain reaction in human and murine skin. The mechanisms regulating LL-37 expression in endothelial cells were examined by gene silencing and chromatin immunoprecipitation. Serum LL-37 levels were determined by enzyme-linked immunosorbent assay. RESULTS: In SSc lesional skin, LL-37 expression was increased in dermal fibroblasts, perivascular inflammatory cells, keratinocytes and, particularly, dermal small vessels. Expression positively correlated with interferon-α expression, possibly reflecting LL-37-dependent induction of interferon-α. In SSc animal models, bleomycin-treated skin exhibited the expression pattern of CRAMP, a murine homologue of LL-37, similar to that of LL-37 in SSc lesional skin. Furthermore, Fli1+/- mice showed upregulated expression of CRAMP in dermal small vessels. Fli1 binding to the CAMP (LL-37 gene) promoter and Fli1 deficiency-dependent induction of LL-37 were also confirmed in human dermal microvascular endothelial cells. In the analysis of sera, patients with SSc had serum LL-37 levels significantly higher than in healthy controls. Furthermore, serum LL-37 levels positively correlated with skin score and the activity of alveolitis and were significantly elevated in patients with digital ulcers compared with those without. CONCLUSIONS: LL-37 upregulation, induced by Fli1 deficiency at least in endothelial cells, potentially contributes to the development of skin sclerosis, interstitial lung disease and digital ulcers in SSc.

Fli1 deficiency contributes to the downregulation of endothelial protein C receptor in systemic sclerosis: a possible role in prothrombotic conditions.

BACKGROUND: Endothelial protein C receptor (EPCR), expressed predominantly on endothelial cells, plays a critical role in the regulation of the coagulation system and also mediates various cytoprotective effects by binding and activating protein C. So far, the role of EPCR has not been studied in systemic sclerosis (SSc). OBJECTIVES: To investigate the potential contribution of EPCR to the development of SSc. METHODS: EPCR expression was examined in skin samples and cultivated dermal microvascular endothelial cells by immunostaining, immunoblotting and/or quantitative reverse-transcription polymerase chain reaction. Fli1, binding to the PROCR promoter, was assessed by chromatin immunoprecipitation. Serum EPCR levels were determined by enzyme-linked immunosorbent assay in 65 patients with SSc and 20 healthy subjects. RESULTS: EPCR expression was decreased in dermal small vessels of SSc lesional skin compared with those of healthy control skin. Transcription factor Fli1, deficiency of which is implicated in SSc vasculopathy, occupied the PROCR promoter, and EPCR expression was suppressed in Fli1 small interfering RNA-treated endothelial cells and dermal small vessels of Fli1(+/-) mice. In patients with SSc, decreased serum EPCR levels were associated with diffuse skin involvement, interstitial lung disease and digital ulcers. Furthermore, serum EPCR levels inversely correlated with plasma levels of plasmin-α2-plasmin inhibitor complex (PIC). Importantly, bosentan significantly reversed circulating EPCR and PIC levels in patients with SSc, and the expression of Fli1 and EPCR in dermal small vessels was elevated in patients treated with bosentan compared with untreated patients. CONCLUSIONS: Endothelial EPCR downregulation due to Fli1 deficiency may contribute to hypercoagulation status leading to tissue fibrosis and impaired peripheral circulation in SSc.

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.

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.

Effects of bosentan on nondigital ulcers in patients with systemic sclerosis.

BACKGROUND: Bosentan is an oral dual endothelin receptor antagonist, which has been shown to be efficacious for preventing new digital ulcers in patients with systemic sclerosis (SSc) in two high-quality randomized controlled trials. However, its efficacy for nondigital ulcers in SSc remains unknown. OBJECTIVES: To evaluate the efficacy of bosentan on nondigital ulcers in patients with SSc. METHODS: Bosentan was administered to five patients with SSc with pulmonary arterial hypertension, who also had nondigital ulcers refractory to conventional treatments. The efficacy of bosentan on nondigital ulcers and its association with clinical features of ulcers were analysed. RESULTS: The nondigital ulcers refractory to conventional treatments were significantly improved by the administration of bosentan in cases surrounded with severe cyanosis. In contrast, nondigital ulcers without cyanosis were still refractory to bosentan therapy. CONCLUSIONS: Bosentan may be efficacious for accelerating the healing of nondigital ulcers with severe cyanosis, suggesting that nondigital ulcers caused by severely impaired peripheral circulation are highly responsive to this treatment.

Hyperviscosity syndrome associated with systemic plasmacytosis.

Systemic plasmacytosis is characterized by plasma cell proliferation in multiple organs including skin, and by polyclonal hypergammaglobulinaemia. Hyperviscosity-related retinopathy has never been described with this condition, to our knowledge. We report a case of systemic plasmacytosis in a 49-year-old Japanese woman, who presented with fever, multiple erythematous plaques, hypergammaglobulinaemia, renal failure and bilateral retinal haemorrhage. Reduction of immunoglobulin with oral steroid reversed the retinopathy related to hyperviscosity syndrome. When marked hypergammaglobulinaemia is found in a patient with systemic plasmacytosis, funduscopic examination should be performed to reveal early asymptomatic retinal changes, because the retinopathy is treatable by control of the underlying disease.

Endothelial cell-binding antibodies in patients with systemic lupus erythematosus.

Abstract The implications of endothelial cell-binding IgG antibodies (EC IgG) in systemic lupus erythematosus (SLE) was evaluated by determining level of EC IgG in sera from 112 SLE patients. The serum EC IgG level was determined by the cyto-ELISA method using human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), and aortic endothelial cells (HAEC) as antigens. The levels of EC IgG were significantly higher among patients with SLE than among healthy control subjects (P < 0.001), and 68% (76/112) of SLE patients were shown to be EC IgG-positive. In patients with active lupus nephritis, the level of EC IgG was statistically and significantly elevated compared with those without lupus nephritis (P < 0.05). Negative correlations between EC IgG level and levels of CH50, C3, and lymphocyte count were revealed (P < 0.05, P < 0.005, and P < 0.05, respectively). When clinical course was evaluated, the levels of EC IgG correlated with disease activity. Definitive correlations in antibody levels between HUVEC and HMVEC, and between HUVEC and HAEC were revealed (both P < 0.0001). The results of this study revealed that the EC IgG in patients with SLE was involved in the onset of clinical manifestation, especially in patients with active lupus nephritis.

A ubiquitin-like system mediates protein lipidation.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.

The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway.

Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.

ASC1/RAS2 suppresses the growth defect on glycerol caused by the atp1-2 mutation in the yeast Saccharomyces cerevisiae.

To better define the regulatory role of the F(1)-ATPase alpha-subunit in the catalytic cycle of the ATP synthase complex, we isolated suppressors of mutations occurring in ATP1, the gene for the alpha-subunit in Saccharomyces cerevisiae. First, two atp1 mutations (atp1-1 and atp1-2) were characterized that prevent the growth of yeast on non-fermentable carbon sources. Both mutants contained full-length F(1)alpha-subunit proteins in mitochondria, but in lower amounts than that in the parental strain. Both mutants exhibited barely measurable F(1)-ATPase activity. The primary mutations in atp1-1 and atp1-2 were identified as Thr(383) --> Ile and Gly(291) --> Asp, respectively. From recent structural data, position 383 lies within the catalytic site. Position 291 is located near the region affecting subunit-subunit interaction with the F(1)beta-subunit. An unlinked suppressor gene, ASC1 (alpha-subunit complementing) of the atp1-2 mutation (Gly(291) --> Asp) restored the growth defect phenotype on glycerol, but did not suppress either atp1-1 or the deletion mutant Deltaatp1. Sequence analysis revealed that ASC1 was allelic with RAS2, a G-protein growth regulator. The introduction of ASC1/RAS2 into the atp1-2 mutant increased the F(1)-ATPase enzyme activity in this mutant when the transformant was grown on glycerol. The possible mechanisms of ASC1/RAS2 suppression of atp1-2 are discussed; we suggest that RAS2 is part of the regulatory circuit involved in the control of F(1)-ATPase subunit levels in mitochondria.