Peg sod

Coelenterazine was purchased from Nanolight (#303). PEG-SOD (pegylated superoxide dismutase #S9549), PEG-CAT (pegylated catalase #C4963), uric acid (#U2625), d-glucose (#G8270), 4-OHCA (alpha-cyano-4-hydroxycinnamic acid #M7033), AOAC (aminooxyacetic acid #C13408), GKA50 (#L2630), Ranolazine (#R6152) and streptozotocin (#S0130) were all purchased from Sigma Aldrich.

Procyanidin B2 (≥90% purity) was obtained from Extrasynthese (Genay, France). Glutamate, glycine, PEG-SOD, Hoechst stain, p-phenylenediamine, and monoclonal antibody against β-tubulin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). HA14-1 (2-amino-6-bromo-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester) and sodium nitroprusside (SNP) were obtained from Calbiochem (San Diego, CA, USA). FITC-conjugated secondary antibody was obtained from Jackson Immunoresearch Laboratories (West Grove, PA, USA). Nitric Oxide Assay kits (EMSNO) were obtained from Thermo Fisher Scientific (Rockford, IL, USA).

Unless stated otherwise, all reagents were from Merck (Darmstadt, Germany). Inhibitors used were: CGS35066, an ECE-1 inhibitor (Tocris Bioscience/Bio-Techne; Wiesbaden, Germany); valsartan, an angiotensin receptor type I (AT1) antagonist (Sigma-Aldrich; Schnelldorf, Germany, Merck, Darmstadt, Germany); PD-184352, an inhibitor of extracellular signal-regulated kinases (ERK) (Cell Signaling Technology, Danvers, MA, USA); and PEG-SOD, a superoxide dismutase-polyethylene glycol conjugate (Sigma-Aldrich^®, Merck). Their effective concentrations were determined in preliminary experiments.

The NAD(P)H oxidase inhibitor VAS2870 and pegylated superoxide dismutase (PEG-SOD) were obtained from Sigma–Aldrich and dissolved in 20% Pluronic F-127 (Sigma–Aldrich) at 250 μg/ml and 1667 units/ml concentrations, respectively. Both solutions were prepared and maintained at 4°C until use. Prior to injury, 300 μl of dissolved VAS2870, PEG-SOD or vehicle were applied perivascularly to the artery and allowed to solidify for 30 min, after which balloon injury was performed. Arterial specimens were collected at 90 min and 30 days post injury for analysis.

The assay was done as described [36] (link). Briefly, cells were plated in 12-well dishes 24 h before the experiment. Cells were washed once with warm PBS, followed by incubation with 300 µL of pre-warmed assay buffer [HEPES-buffered Tyrode's solution (no FBS) containing 50 µM Amplex Red (Invitrogen) and 2 U/mL Horse Radish Peroxidase (Sigma)] containing 10 µM LPC 18:1 or PBS (vehicle) for 15 min. Polyethylene glycol catalase (PEG-catalase) 300 U/mL or polyethylene glycol superoxide dismutase (PEG-SOD) 75 U/mL (both Sigma) were added in order to detect catalase-sensitive peroxides and superoxide radicals in the medium. The buffer was then transferred to a black 96 well plate and fluorescence was measured at excitation and emission wavelengths of 540 and 580 nm, respectively. Relative fluorescence units were normalised to cellular protein concentration. Final values represent catalase sensitive H₂O₂ values obtained by subtration of values measured in the presence of catalase from values obtained upon measurements in the absence of catalse.