Ascorbic acid

Primary human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HUVECs were cultured and grown in vascular cell basal medium (PCS‐100‐030; ATCC) supplemented with bovine brain extract (0.2%), recombinant human (rh) EGF (5 ng/ml), L‐glutamine (10 mM), heparin sulphate (0.75 IU/ml), hydrocortisone hemisuccinate (1 μg/ml), 2% FBS and ascorbic acid (50 μg/ml). All supplements were purchased from ATCC (#PCS‐100‐040). These cells were cultured at 37°C in a CO₂ humidified atmosphere and detached from the growth plates using non‐enzymatic cell dissociation solution (Cellstriper; Corning Costar).

Human umbilical vein endothelial cells (HUVECs) were cultured in 12 well-plates (SPL Life Sciences, Pocheon, Korea) in a vascular cell basal medium (ATCC, Manassas, VA, USA). The culture medium comprised 0.2% bovine brain extract (BBE; ATCC), 5 ng/mL recombinant human epidermal growth factor (rhEGF; ATCC), 10 mM L-glutamine (ATCC), 0.75 Units/mL heparin sulfate (ATCC), 1 µg/mL hydrocortisone (ATCC), 50 µg/mL ascorbic acid (ATCC), 2% fetal bovine serum (ATCC), and 1× antibiotic-antimycotic agents (Thermo Fisher Scientific), and culturing was performed in a 5% CO₂ incubator at 37 °C. Confluent cells were detached using 0.05% trypsin EDTA (Thermo Fisher Scientific) in Hank’s balanced salt solution (HBSS; Hyclone, Logan, UT, USA). For recombinant hADPN treatment, HUVECs under 10 passages were grown until 80−90% confluency, then treated with 10 µg/mL of EW-derived recombinant hADPN, HEK293 cell-derived commercial recombinant hADPN (Biovendor R&D), or Hi-5 cell-derived commercial recombinant hADPN (PeproTech) for 7 days. After treatment with recombinant hADPN, cells were subjected to stress induced by 100 µM H₂O₂ for 1 h.

Primary human aortic endothelial cells (HAECs) and vascular cell basal medium supplemented with VEGF (5 ng/mL), EGF (5 ng/mL), FGF (5 ng/mL), IGF-1 (15 ng/mL), L-glutamine (10 mM), heparin sulfate (0.75 U/mL), hydrocortisone (1 µg/mL), ascorbic acid (50 µg/mL), fetal bovine serum (FBS), penicillin (10,000 U/mL), streptomycin (10 mg/mL) and amphotericin B (25 µg/mL) were all obtained from American Type Culture Collection (ATCC, Manassas, VA). Acetylated PGP and PGG were purchased from Bachem (Torrance, CA) and purified to neutrophil chemotactic activity. Human IL-8 with carrier was obtained from Cell Signaling Technology (Danvers, MA). Finally, the selective CXCR2 antagonist SB225002 was purchased from Sigma Aldrich (St. Louis, MO).