2.7. Endothelial Cell Apoptosis Assay

The assay was performed as described previously (Jimenez et al., 2000). Briefly, primary human dermal microvascular EC (PromoCell-Fisher Scientific) were grown in Endothelial Growth Medium MV (EGM, Fisher Scientific), supplemented with 5% FBS and growth factors (EGM Bullet Kit, Fisher Scientific) and used between passages 3 and 5. For apoptosis induction, EC were cultured overnight in 1% FBS and treated for 48 hours with an increasing concentration of sterile filtered peptides, alone (1–100 nM range) or in combination with VEGF (10 ng/mL). Apoptosis was detected by using terminal deoxynucleotidyl transferase dUTP by nick-end labeling (TUNEL) assay kit according to the manufacturer’s instructions (Roche, Indianapolis, IN). The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Apoptosis was visualized by fluorescence microscopy (Nikon, Diaphot 2000). Random images were taken at 20x magnification and quantified using Nikon Elements software. Apoptotic fraction or % apoptosis was determined in at least 10 images per condition and the average values calculated. Alternatively, immunostaining for cleaved, active Caspase-3 was used to detect apoptotic cells. Statistical significance was determined using one-way ANOVA.