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.