Fucosylated chondroitin sulfates from the body wall of the sea cucumber <i>Holothuria forskali</i>. Conformation, selectin binding and biological activity

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: →3)GalNAcβ4,6S(1→4) [FucαX(1→3)]GlcAβ(1→, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Lex blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu2+-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention

The Role of Heparanase in Pulmonary Cell Recruitment in Response to an Allergic but Not Non-Allergic Stimulus

Heparanase is an endo-a-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa(-/-)) mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa(-/-) and wild type (WT) control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa(-/-) mice displayed a significantly reduced eosinophil (but not neutrophil) recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD) found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases

The effect of genetic modification of heparanase on neutrophil recruitment to the lung.

<p>The total number of inflammatory cells (A, C) and neutrophils (B, D) recruited to the lung following acute exposure of wild-type (WT), and Hpa^-/- mice to intranasal (i.n.) saline, zymosan (A, B) or LPS (C, D). Data presented as box plots (median, 25–75 percentile) with whiskers representing 5–95% confidence interval. The number of animals represented by box plots in each panel is as follows; Panel A, B: Saline treated group (WT; n = nil ♀, 5 ♂; Hpa^-/-, n = 2 ♀, 1 ♂) and zymosan treated group (WT; n = 4 ♀, 4 ♂; Hpa^-/-, n = 3 ♀, 4 ♂). *p < 0.05 compared with saline; †No significant difference compared with WT zymosan. Panel C, D: Saline treated group (WT: wild type; n = 2 ♀, 3 ♂; Hpa^-/-, n = 3 ♀) and LPS treated group (WT; n = 5 ♀; Hpa^-/-, n = 4 ♀). *p < 0.05 compared with saline; †No significant difference compared with WT LPS. Data obtained from one experiment.</p

Effect of heparanase gene deletion on allergic inflammation induced by ovalbumin (OVA).

<p>Box plots showing (A) total cell, (B) eosinophil, (C) macrophages, (D) lymphocytes, (E) neutrophils in bronchoalveolar lavage fluid and (F) OVA specific IgE in serum in control and OVA immunized and challenged mice. Mice were sensitized by repeated i.p. injections with 10 μg OVA or were non-sensitized (controls). All mice received three i.n. instillations with 20 μg OVA and lavage was undertaken 24 h after the last intranasal administration of OVA. Data expressed as blox plots (median, 25–75 percentile) with whiskers representing 5–95% confidence interval (7–12 animals for cell data and 6–10 for IgE data). *p < 0.05 compared with control; †No significant difference compared with Hpa^-/- control. Data pooled from four independent experiments.</p

Heparanase expression in lung tissue from normal subjects and COPD patients of varying severity.

<p>Each point represents the intensity of staining for heparanase expression in normal human lung tissue and in subjects with varying severities of COPD (A). Representative images are shown (B-F reflecting Gold 0–1, Gold 2, Gold 3–4, healthy subject, and isotype control, respectively. Red: heparanase; blue: DAPI (DAPI not performed for isotype control). Image brightness was uniformly adjusted for clarity and all heparanase quantification was performed using unadjusted (raw) images. Magnification was 40 x for all images. The data was analyzed as continuous variables and there was a significant linear trend (post-ANOVA, r-squared = 0.27 and p = 0.029). Horizontal line represents mean and limits represent SEM.</p

The effect of pharmacological treatment with the heparanase inhibitor Muparfostat on neutrophil recruitment to the lung.

<p>The total number of inflammatory cells (A, C) and neutrophils (B, D) recruited to the lung following acute exposure to zymosan (A, B) or LPS (C, D). Wild-type (WT), and Hpa^<b>-/-</b> mice received intranasal (i.n.) saline, zymosan (A, B) or LPS (C, D). The number of total cells (A) and neutrophils (B) in bronchoalveolar lavage fluid 24 h after intranasal administration of zymosan in BALB/c mice treated with Muparfostat (30 mg/kg s.c.) dosed 30 minutes before (q.d.) and 6 h after (b.i.d) zymosan exposure, n = 4 per group. Data presented as box plots (median, 25–75 percentile) with whiskers representing 5–95% confidence interval. *p < 0.05 compared with saline. Data obtained from one experiment. In other experiments total number of cells (C) and neutrophil (D) recruitment to the lung was measured following intranasal administration of LPS in vehicle and Muparfostat (b.i.d.) treated mice (n = 3–9). Data presented as box plots (median, 25–75 percentile) with whiskers representing 5–95% confidence interval. *p < 0.05 compared with saline. Treatment groups were not significantly different from vehicle (symbol not reported). Data was obtained from two independent experiments.</p