Measurements of gene expression levels by quantitative real-time PCR

Messenger RNA (mRNA) expression levels of E-selectin, intracellular adhesion molecules (ICAM-1), FMS-like tyrosine kinase 1 (FLT-1), kinase insert domain receptor (KDR), angiopoietin-2 (ang-2), and von Willebrand factor (vWF) were determined by qPCR similarly to the method described in our previous studies [20–22]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a house-keeping gene. HUVECs were seeded in 24-well plates similar to MTT experiments and stimulated in FBS-free ECM with e-TRAP, synthetic TRAP, or EMD at concentrations of 10 and 50 μg/ml. Cells stimulated with FBS-free ECM supplemented with 0.001 % of acetic acid served as vehicle control. Isolation of total cellular mRNA and transcription into cDNA was performed using the TaqMan Gene Expression Cells-to-CT kit (Ambion/Applied Biosystems, CA, USA) according to manufacturer’s instructions. Real-time PCR was performed on an Applied Biosystems Step One Plus real-time PCR instrument (Applied Biosystems, CA, USA) using TaqMan® gene expression assays with the following ID numbers (all from Applied Biosystems, CA, USA): E-selectin, Hs00174057_m1; ICAM-1, Hs00164932_m1; FLT-1, Hs01052961; KDR-1, Hs00911700_m1; ang-2, Hs01048043_m1; vWF, Hs00169795_m1; GAPDH, Hs99999905_m1). Duplicate PCR reactions were prepared and the point at which the PCR product was first detected above a fixed threshold (termed cycle threshold, C_t), was determined. Changes in the expression of target genes were calculated using 2^−ΔΔCt method, where ΔΔC_t = (C_t ^target − C_t ^GAPDH)_sample − (C_t ^target − C_t ^GAPDH)_{vehicle control}.