The sulfated polysaccharides DS500 and fucoidan interact with NRP1 and reduce cell-surface levels of NRP1 on endothelial cells. (A) Effects of polysaccharides on NRP1 binding to heparin. NRP1 (20 nM) was passed over a heparin-coated sensor chip without (black line), or with 0.1 μg/mL (blue line) or 1 μg/mL (red line) polysaccharide. (B) Binding of NRP1 to dextran sulfate. NRP1/Fc (1 μg/mL) or gp130/Fc (1 μg/mL) was incubated with heparin-gel, DS-gel, and maltose-gel and the precipitates were detected by anti-Fc antibody. NRP1/Fc or gp130/Fc (100 ng) was loaded as a control. (C) Modulation of cell-surface NRP1 by polysaccharides. HUVECs were incubated with the polysaccharides (0-64 μg/mL, 37°C, 1 hour). After cell washing (1 M NaCl), NRP1 was detected by flow cytometry. Results reflect the relative mean fluorescence intensities with and without polysaccharide. (D) Effects of DS500 on levels of cell-surface molecules NRP1, NRP2, VEGFR-1, VEGFR-2, CD31, VE-cadherin, gp130, and CXCR4. HUVECs were incubated (37°C, 1 hour) with or without DS500 (8 μg/mL). Shaded graphs reflect control staining. (E) Temperature-, concentration-, and time-dependent reduction of cell-surface NRP1 and NRP2 by DS500. HUVECs were incubated with DS500. NRP1 and NRP2 were detected by flow cytometry. (F) HUVECs were treated (37°C, 1 hour) with or without DS500 (8 μg/mL), stained for NRP1 (green) and DAPI (blue), fixed, and observed through an Olympus IX51 phase-contrast microscope equipped with a 10×/0.25 PhC objective lens and a 10× eyepiece (Olympus Optical, Melville, NY), and were photographed with a Retiga 1300 digital camera (Qimaging, Burnaby, BC). Original magnification ×100. (G) NRP1 detected on HUVECs incubated with DS500 (0-8 μg/mL, 37°C, 1 hour) in the presence of 1% or 95% human serum.