Search

Systemic sclerosis (SSc) is an autoimmune and vascular disease resulting in multiple organ fibrosis, in which IL-6 and T helper (Th)2/Th17 cytokines serve as critical disease drivers. LIGHT is a proinflammatory cytokine promoting IL-6 production in lung fibroblasts and Th1 chemokine expression in dermal fibroblasts (DFs) stimulated with IFN-γ. In this study, we investigated the potential contribution of LIGHT to SSc development using clinical samples and animal models. In SSc-involved skin, LIGHT was upregulated in inflammatory cells, whereas herpesvirus entry mediator (HVEM), a receptor of LIGHT, was downregulated in DFs. Similar expression profiles of LIGHT and HVEM were reproduced in bleomycin-treated mice. Transcription factor FLI1 bound to the HVEM promoter, and FLI1 small interfering RNA suppressed HVEM expression in normal DFs. In SSc DFs, LIGHT significantly increased IL-6 production, whereas IFN-γ/LIGHT-dependent Th1 chemokine induction was decreased compared with that in normal DFs. Importantly, LIGHT small interfering RNA significantly attenuated bleomycin-induced skin fibrosis, and serum LIGHT levels were elevated in patients with diffuse cutaneous SSc and positively correlated with clinical parameters reflecting skin and pulmonary fibrosis. Taken together, these results suggest that altered response of DFs to LIGHT, namely increased IL-6 production and decreased Th1 chemokine expression, contributes to the development of skin fibrosis in SSc., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Intravenous cyclophosphamide pulse, a standard treatment for systemic sclerosis (SSc)-related interstitial lung disease, elicits a disease-modifying effect on SSc vasculopathy, such as fostering microvascular de-remodeling. To investigate the molecular mechanism by which cyclophosphamide mitigates SSc vasculopathy, we employed endothelial cell-specific Fli1 knockout mice that mimic the functional and structural vascular abnormalities characteristic of SSc. Biweekly cyclophosphamide injection improved vascular permeability and structural abnormalities of endothelial cell-specific Fli1 knockout mice in 2 weeks and in 3 months, respectively. In endothelial cell-specific Fli1 knockout mice, a single dose of cyclophosphamide was sufficient to normalize the decreased expression of α-smooth muscle actin in dermal blood vessels and improve the impaired neovascularization in skin-embedded Matrigel plug. Under the same condition, the decreased expression of vascular endothelial cadherin, platelet-derived growth factor B, S1P 1 , and CCN1 (molecules associated with angiogenesis and/or vasculogenesis) was reversed along with the reversal of endothelial Fli1 expression. In SSc patients, serum CCN1 levels were significantly increased after intravenous cyclophosphamide pulse. Taken together, these results indicate that cyclophosphamide improves Fli1 deficiency-dependent vascular changes by normalizing the expression of angiogenesis- and vasculogenesis-related molecules and endothelial Fli1, which may help to explain the beneficial effect of cyclophosphamide on SSc vasculopathy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Two forms of alpha-fucosidase from newborn rat epidermis were separated by gel filtration on Sephadex G-150. These enzymes termed, fucosidase I which was eluted in the void volume and fucosidase II (molecular weight approximately 50,000). Both enzymes had pH optima for 4-methylumbelliferyl-alpha-L-fucoside hydrolysis between 5.5-6.5. Km values for fucosidase I and II with the same substrate were 3.7 X 10(-5) and 5.4 X 10(-5) M, respectively. Three different forms of new born rat epidermal alpha-fucosidase were separated by isoelectric focusing. Evidence is present which indicates that the electrophoretic heterogeneity of alpha-fucosidase is due in part to the binding of sialic acid to the primary gene product.

Two forms of beta-galactosidase from newborn rat epidermis could be separated by DEAE-cellulose chromatography. Both enzymes showed similar enzymic properties. They had a pH optimum around 3.5--4.5 and the optimal temperature of these enzymes was approximately 60 degrees C. They were not affected by divalent cations, ethylenediaminetetraacetic acid(EDTA) and 2-mercaptoethanol(2-ME), while rho-chloromercuribenzoic acid (PCMB) was a strong inhibitor for each enzyme. These enzymes showed the same Km value (1.25 x 10(-4) M) towards 4-methylumbelliferyl-beta-D-galactoside. However they had different isoelectric points at pH 6.3 and 9.0, respectively. Six different forms of beta-galactosidase activity were found by using isoelectric focusing. When the crude extract was incubated with neuraminidase before electrofocusing, the acidic forms of the enzyme were largely lost and converted to more basic forms without loss of the total activity. This finding suggests the glycoprotein nature of newborn rat epidermal beta-galactosidase.

Isoelectric focusing and concanavalin A-Sepharose chromatography were used to study the multiple forms and glycoprotein natures of so-called lysosomal hydrolases from psoriatic scales. Acid phosphatase appeared as 5 different forms with pI values of 6.5, 6.1, 5.8, 5.6 and 5.45. Seven isoenzymes of alpha-fucosidase were identified with pI values of 6.4, 6.2, 5.9, 5.75, 5.65, 5.4 and 5.2. Acid alpha-mannosidase activity appeared as one peak with pI value of 6.75 and a weak activity of neutral alpha-mannosidase was present with pI value of 6.7. Incubation of the extract with neuraminidase increased their pI values of acid phosphatase, alpha-fucosidase and alpha-mannosidase to the more basic forms. This finding suggests that epidermal acid phosphatase, alpha-fucosidase and alpha-mannosidase have some N-acetylneuraminic acid residues. In addition concanavalin A-Sepharose column chromatography was also performed to confirm the glycoprotein nature of acid phosphatase. This enzyme was bound to the column and not released from the column even with the treatment of 0.5 M NaCl, but the enzyme was eluted from the column with the treatment of alpha-methyl-D-glucoside.