Growth Factors-induced Tubular Morphogenesis is Down-regulated by the MEK Inhibitor U0126 in Cultured Human Endothelial Cells

Abstract

Abstract: : Purpose: In vitro studies were performed in cultured human umbilical vein endothelial cell line (HUVEC) to test the possible involvement of IP3K and MEK in growth factors-induced tubular morphogenesis. Methods: To evaluate tube and cell aggregate formation, HUVECs (PromoCell, Germany) were seeded (7X10⁵/well) in 24 well microplates coated with Matrigel (Becton Dickinson, Bedford, MA) and covered by ECBM containing supplement with growth factors (EDF, bFGF, IGF-1, PDGF, and TGF-beta; 5 pg/ml - 5 ng/ml). Four hours later, the medium was replaced with a fresh preparation containing the test compound or its vehicle (DMSO 0.001%-0.01%). Cells without any test compound belonged to "baseline" control. Cell proliferation rate was determined by a BrdU kit (Boehringer-Mannheim, Germany). For the follow-ups of NF-ΚB-DNA-binding and COX-2 expression, HUVECs were seeded on collagen Type 1 and the medium contained the same concentration of growth factors as that used for the tube formation studies. Results: The MEK-1 inhibitor, U0126 (Promega) (0.1 –1 µM) caused a significant inhibition of tube formation characterized by the length of the tubes and the area of tight cellular aggregates (p<0.001 for both effects). The IP3K inhibitor, LY294002 (Calbiochem) did not exert any significant inhibitory effect on tubular morphogenesis using the same dose-range. Neither of the compounds tested altered cell proliferation rate. Nuclear protein extracts (NPXTs) and total RNA were prepared and gel shift assays were performed using NPXTs as transcription factor source. Relative COX-2 and VEGF RNA message levels were determined by RT-PCR. NF-ΚB-DNA binding and COX-2 expression was substantially inhibited by the U0126 compound.Conclusions: These data suggest that the growth factor-induced signal transduction includes MEK as a powerful factor but does not seem to need IP3K to stimulate neovascularization.

Keywords: hypoxia • gene/expression • growth factors/growth factor receptors