Notch1-induced HUVEC morphological changes were enhanced by the VEGFR-1-specific ligand PlGF. HUVEC were transduced with Ad-LacZ or Ad-N1IC (MOI 40) and cultured on collagen type I gels in serum free medium with or without 50 ng/ml PlGF and evaluated for the formation of cellular extensions. Representative images from each cell culture are shown (10× magnification). (A-B) Ad-LacZ HUVEC remained a homogenous monolayer in the absence (NT) or presence of PlGF. (C) In the absence of PlGF, Ad-N1IC HUVEC undergo morphological differentiation characterized by one or two extensions per cell (black arrowheads). (D) In presence of PlGF, there is an increase number of Ad-N1IC HUVEC undergoing morphological changes and the number of extensions per cell (open arrowheads). (E) Quantification of total number of Ad-N1IC HUVEC with cellular extensions and cells with multiple (>3) processes with or without PlGF. Number of cells with cellular extensions were counted per 10× field, for five separate fields. Data is representative of the mean plus or minus SD of three separate experiments. * P < 0.01 compared with cultures without PlGF. (F) RT-PCR of RNA isolated from Ad-LacZ and Ad-N1IC HUVEC for VEGF-A (22 cycles), PlGF (25 cycles) and GAPDH (22 cycles), as a control.