Heparin

Freshly isolated HUVEC were maintained in endothelial cell growth medium (EGM) supplemented with SingleQuots (EGM BulletKit CC-3124, Lonza). They were then plated in a 96-well plates (14 × 10³ cells/well) previously coated with growth factor-depleted Matrigel (Becton-Dickinson, Bedford, MA) in M199 medium supplemented with 20% FBS, 1% glutamine, 1% antibiotic–antimycotic solution (Biological Industries) and 50 U/100 ml heparin (Biomedical Technologies Inc., Stoughton, MA). Cells were treated with trT2-50m (2 μM), peptides or PBS, in addition to angiogenin (R&D Systems, Inc. Minneapolis, MN), or VEGF (Protein Laboratories, Rehovot, Israel) (1 μg/ml each). After 8 h of incubation at 37°C, the plates were photographed and the extent of tube formation was counted using Image J (NIH, Bethesda, MD) software. Five individual determinations were performed for each treatment. Statistical analysis was performed using one way ANOVA for multiple comparison and 2-sample t-test.

Freshly isolated HUVEC were maintained in endothelial cell growth medium (EGM) supplemented with SingleQuots (EGM BulletKit CC-3124, Lonza). They were then plated in a 96-well plate (14 × 10³ cells/well) previously coated with growth factor-depleted Matrigel (Becton-Dickinson, Bedford, MA) in M199 medium supplemented with 20% FCS, 1% glutamine, 1% antibiotic–antimycotic solution (Biological Industries) and 50 U/100 ml heparin (Biomedical Technologies Inc., Stoughton, MA). Cells were simultaneously treated with either trT2-50 (2 μM) or PBS, in addition to angiogenin (R&D Systems, Inc. Minneapolis, MN), or VEGF (Protein Laboratories, Rehovot, Israel) (1 μg/ml each). After 8 h of incubation, at 37°C, the plates were photographed, and the extent of tube formation was counted using Image J (NIH, Bethesda, MD) software. Five individual determinations were performed for each treatment.

HUVEC were isolated from umbilical cords obtained from the maternity unit at Meir Medical Center, Kfar Saba, Israel [13 (link)]. The Ethical Review Committee approved the study and the parturient provided written informed consent. HUVEC were identified by their typical cobblestone morphology and by immunostaining for von Willebrand factor. HUVEC were grown in M-199 medium supplemented with 20% FCS, 100 U/ml penicillin, 100 μ/ml streptomycin (Biological Industries, Bet Haemek, Israel), 5 U/ml heparin, and 25 μ/ml endothelial mitogen (Biomedical Technologies Inc., Stoughton, MA, USA). Confluent cultures of HUVEC were used for experiments at passages 3–4. The cells were stimulated in media containing 200 μg/μl HSA and 100 mg/dl glucose (control group) or 200 μg/μl AGE-HSA and 250 mg/dl glucose (diabetic-like environment) for 24 h. Calcitriol 10^-10 mol/l (PH&T SpA, Milan, Italy), corresponding to physiological blood concentrations, [16 ,17 ] was added to cells 1 h after having started the stimulation with AGE-HSA and glucose for an additional 23 h.