Figure 2

Inhibition of the ERK MAP kinase in the presence of H ₂ O ₂ is associated with an increase of endothelial permeability. A) Exponentially growing HUVECs were pretreated with vehicle (0.25% DMSO) or with PD098059 (50 μM) for 60 minutes. Thereafter, HUVECs were treated or not with H₂O₂ (250 μM) for 30 minutes. Proteins were extracted, were separated by SDS-PAGE, and were transferred onto nitrocellulose membrane. Inhibition of the ERK pathway was revealed by western blot using phospho-ERK specific antibody (upper panel). Total ERK (ERK2) and tubulin-α expression levels were determined as loading controls (middle and lower panels, respectively). A representative blot is shown B) HUVECs were plated at a density of 0.6×10⁵ per well in the upper part of a Boyden chamber and were cultivated for 3 days until the formation of a tight monolayer. Then, the monolayers were pretreated with vehicle (0.25% DMSO) or with PD098059 (50 μM) for 60 minutes, and then they were treated or not with H₂O₂ (250 μM) for 30 minutes. Passage of FITC-labelled dextran through the monolayer of HUVECs was measured immediately after treatment. The results represent the permeability changes obtained in a representative experiment and are expressed as the mean fold increases (± SEM) in treated cells relative to untreated cells (n=3). p value was determined by using unpaired Student t test.