Methods
Human umbilical vein ECs (HUVECs, pooled, Lonza) or human coronary artery EC (HCAECs, Lonza) were cultured in EC growth medium-2 (EGM™-2 Bulletkit™; Lonza) containing growth factors or MCDB 131 (Gibco) supplemented with serum and antibiotics. Following 60-70% confluence, cells were starved over-night in 1% FBS and then treated with 5, 10 and 20mM of VPA (Santa Cruz Biotechnology) for 24 hours. Control group were treated with the diluent. RNA were extracted using Trizol (Invitrogen) and cDNA was synthesized (Quantitect, Qiagen) according to manufacturer’s instruction. Quantitative (q)PCR was performed to measure expression level of genes using SYBR® Select Master Mix (Applied Biosystems) and primers for ACE-2[20] IL-6 (Forward - 5′-CATTGGAGCAAGTGTTGGATCTT-3′ and Reverse - 5′-GAGCTAATGCATGCCATTCTCA-3′), and inter-cellular adhesion molecule-1 (ICAM-1)[20] in QuantStudio™ 3 Real-Time PCR System. GAPDH was used as internal control.[20] Differences between two groups were calculated using Student’s T-test and differences between more than two groups were calculated using one-way ANOVA with Tukey’s test. A p-value of <0.05 was considered significant.