Selective inhibitory effects of L.s.-P and its fractions L.s.-1.0 and L.s.-1.25 on FBS-induced HUVEC tubulogenesis.

<p>(<b>A</b>) Representative photographs of HUVEC cultured on Matrigel in the presence of FBS along with 100 µg/ml of indicated parent fucoidan or purified fractions. (<b>B</b>) Quantitative analysis of tube-like structures <i>in vitro</i> using three different polysaccharide concentrations. Analysis was obtained by counting closed areas (tubes) in at least four different fields. Data are collected from at least three independent experiments. All data were expressed as the percentage of tubes/cm² of treated cells <i>vs</i> control: filled, open and grey bars indicate the effect induced by 100, 10 or 1 µg/ml polysaccharides respectively.</p

Effect of polysaccharide preparations on tumor growth and angiogenesis <i>in vivo</i>.

<p>C57BL/6 (B6) mice were injected with 500 µl of Matrigel containing 1×10⁵ B16-F10 cells in PBS or 100 µg of a non-fractionated polysaccharide mixture <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b>. After 6–7 days, tumors were removed and hemoglobin content was evaluated by using the Drabkin colorimetric method. Results are expressed as the amount of hemoglobin (mg)/Matrigel weight (mg) (<b>A</b>) (**<i>P</i><0.01). (<b>B</b>) Flow cytometry analysis of the frequency of CD34⁺ endothelial cells on Matrigel plugs embedded with B16 melanoma cells. (**<i>P</i><0.01) (<b>C</b>) <i>In vitro</i> cell growth of B16 melanoma cells exposed to 100 µg/ml of <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b>. Data are the mean ± SEM of three independent experiments. (<b>D</b>) B6 mice were injected with 500 µl Matrigel containing 1×10⁵ B16-F10 cells. <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b> were injected <i>i.p.</i> at doses of 50 mg/kg every 3 days and compared to control (PBS). Tumors were removed on day 21 post-implantation, photographed (<b>D</b>) and analyzed for CD31⁺ associated blood vessels (<b>E</b>), microvessel density (<b>F</b>) and weight (<b>G</b>). (*<i>P</i><0.05; **<i>P</i><0.01).</p

Sulfate content, anti-inflammatory and anti-coagulant activities of the polysaccharide preparations from <i>L. saccharina</i> brown seaweed.

a<p>Six animals received 0.9% (wt/vol) NaCl instead of polysaccharides.</p>b<p>The anti-inflammatory activity was determined as the effect on PMN transmigration to the peritoneal cavity of rats (six animals in each group, for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017283#s3" target="_blank"><i>Results</i></a>). The polysaccharide preparations were injected intravenously with a dose of 1.0 and 4.0 mg/kg of the rat weight. Data are presented as mean ± SEM. N is the number of rats in the group.</p>c<p>Anticoagulant activity was measured as the APTT related to a heparin standard with an activity of 140 U/mg. Data are shown as mean ± SEM; N = 4.</p

Effect of L.s.-P and its fractions L.s.-1.0 and L.s.-1.25 on PMN adhesion to platelet-coated surface under flow conditions.

<p>Polysaccharide fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b> or a parent mixture <b>L.s.-P</b> were added at a final concentration of 100 µg/ml to a platelet-coated surface and incubated for 10 min at RT. The same concentration of each compound was also added to non-treated (filled bars) or IB4-pretreated (grey bars) PMN suspensions before addition to platelets. Under the flow conditions, migration of PMN was monitored and photographed. Analysis was performed by counting the number of attached PMN per field in at least 4 different fields. The mean percentage ± SEM with respect to control of at least three independent experiments is represented. *<i>P</i><0.05; **<i>P</i><0.01.</p

Putative structures of the main components of the fractions L.s.-1.0 (A) and L.s.-1.25 (B) obtained from the total sulfated polysaccharide preparation L.s.-P extracted from <i>L. saccharina</i>.

<p>Putative structures of the main components of the fractions L.s.-1.0 (A) and L.s.-1.25 (B) obtained from the total sulfated polysaccharide preparation L.s.-P extracted from <i>L. saccharina</i>.</p