Objective To examine the effect of angiotensin Ⅱ (Ang Ⅱ) on nuclear factor-kappa B (N F-κB) activation in human endothelial cell line ECV304 and the molecular mechanism by which Ang Ⅱ activat es NF-κB. Methods ECV304 cells were transiently cotransfected with an NF-κB/ luciferase reporter gene and inactive NF-κB-inducing kinase (NIK), IκB kinase α (IKK_α), I κB kinase β (IKK_β) mutants or vectors, respectively. The effect on NF- κB was detected by using an electrophoretic mobility shift assay (EMSA) and overex pression 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, d efinite cytoplasmic-to-nuclear translocations of NF-κB activation were detec ted using subunits p50 and p65 induced by Ang Ⅱ. Results The translocation of p50 in nuclei was highly remarkable 2 hours after Ang Ⅱ st imulation, and the activity was somewhat reduced 6 hours after stimulation to th e 18th hour. Northern blot also showed PDGF-B mRNA increased by stimulation of Ang Ⅱ for 18 hours. Conclusion Ang Ⅱ is effective in stimulating NF-κB activation through a pathway dependen t on NIK, IKK_α and IKK_β, and induces PDGF-B transcription in the endothel ial cell line, ECV304.
