Heparan sulfate

Murine BaF3 lymphocytes expressing murine FGFR1IIIc were a gift from Professor David Ornitz (Washington University, St. Louis, USA). Cells were maintained in RPMI-1640 supplemented + 10% FCS + 100 Units/ml penicillin G + 100 μg/ml streptomycin sulfate (all from ThermoFisher, UK) and 1 ng/ml murine interleukin-3 (R&D Systems, UK) at 10⁵ to 10⁶ cells/ml at 37 °C, 5% CO₂. For assays, cells were plated at 10⁵ cells/ml in 96-well, flat bottomed Costar tissue culture plates (Corning, USA) in 100 μl growth medium without interleukin-3 supplemented with FGF-2 (R&D Systems, UK) and heparan sulfate at the indicated concentrations. Cells were incubated for 72 h at 37 °C, 5% CO₂. Thiazolyl blue tetrazolium bromide (Sigma, UK) was then added to a final concentration of 250 μg/ml and the cells were incubated a further 4 h at 37 °C, 5% CO₂. The assay was stopped with the addition of 50 μl 10% SDS, 0.01 N HCl and the plates were incubated 4 h to overnight at 37 °C to dissolve the formazan product. Plates were read at 570 nm using a Thermo multiskan EX plate reader (ThermoFisher, UK).

Plasma was frozen within 2 hours of collection, and later tested using commercial assays for inflammatory and endothelial biomarkers. Ferritin and IL-6 were measured using the Elecsys electrochemiluminescence immunoassay on the Cobas e 411 analyzer (Roche system). The following biomarkers were analysed using a multiplexed magnetic bead assay on a Luminex 200 platform according to the manufacturer’s specifications: Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), soluble Tie-2, Vascular Cell Adhesion Molecule 1 (VCAM-1), Endocan and atrial natriuretic peptide (ANP) (R&D systems). The following biomarkers were analysed by ELISA: hyaluronan (Cat. No: DHYAL, R&D systems), heparan sulfate (Cat. No: abx513537; Abbexa) and syndecan-1 (SDC-1, Cat. No: 950.640.91; Diaclone), in accordance with the manufacturer’s specifications.