OBJECTIVETo examine the effect of angiotensin II (Ang II) on nuclear factor-kappa B (NF-kappaB) activation in human endothelial cell line ECV304 and the molecular mechanism by which Ang II activates NF-kappaB.

METHODSECV304 cells were transiently cotransfected with an NF-kappaB/luciferase reporter gene and inactive NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKK(alpha)), IkappaB kinase beta (IKK(beta)) mutants or vectors, respectively. The effect on NF-kappaB was detected by using an electrophoretic mobility shift assay (EMSA) and overexpression of the mutants enabled blocking of reporter gene activation induced by Ang II. With immunofluorescence and immuno-electronic microscope techniques, including confocal microscopy and gold particle labeled electronic microscopy, definite cytoplasmic-to-nuclear translocations of NF-kappaB activation were detected using subunits p50 and p65 induced by Ang II.

RESULTSThe translocation of p50 in nuclei was highly remarkable 2 hours after Ang II stimulation, and the activity was somewhat reduced 6 hours after stimulation to the 18th hour. Northern blot also showed PDGF-B mRNA increased by stimulation of Ang II for 18 hours.

CONCLUSIONAng II is effective in stimulating NF-kappaB activation through a pathway dependent on NIK, IKK(alpha) and IKK(beta), and induces PDGF-B transcription in the endothelial cell line, ECV304.v