Materials and methods Materials

Human purified or recombinant TGF-β1, recombinant human or mouse VEGF, goat anti-human VEGFR2 and VEGFR1 antibodies and rabbit neutralizing polyclonal antibodies to human or mouse VEGF were purchased from R&D Systems (Minneapolis, MN); z-VAD-(OMe)-FMK and DMSO from Sigma-Aldrich (St. Louis, MO). Polyclonal antibody to cleaved human caspase 3 was purchased from Cell Signaling Technologies (Beverly, MA); polyclonal antibody to total ERK-2 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-mouse CD31 antibody from Pharmingen (San Jose, CA); mouse and rabbit non-immune IgG from Sigma-Aldrich, and human recombinant FGF-2 from Gibco BRL, Life Technologies, Inc., (Rockville, MD). Neutralizing monoclonal antibody to human FGF-2 (MAb 354FI) was a generous gift from Texas Biotechnology, Inc. (Houston, TX, USA).

Cells and Media

Bovine capillary endothelial cells (BCE) were isolated as described (Seghezzi et al., 1998) and grown in alpha modified minimum essential medium (αMEM; Fisher, Pittsburgh, PA, USA) supplemented with 5% donor calf serum (DCS) and L-glutamine 2 mM (Gibco BRL, Life Technologies). These cells were used between passage 6 and 15 in culture. Human umbilical vein endothelial (HUVE; Clonetics, San Diego, CA) were grown in endothelial cell basal medium-2 (EBM2; Clonetics) containing 2% fetal calf serum (FCS) and the endothelial cell growth supplements provided by the company. HUVE cells were used between passage 3 and 5 in culture. For the apoptosis assays confluent BCE or HUVE cells were starved overnight in their respective medium supplemented with 0.5% DCS or FCS, respectively, after which TGF-β1 (1 ng/ml) was added to the medium and incubation was continued for the indicated time. The time of addition of TGF-β1 was considered as time 0.

Tunel assay for apoptosis

Endothelial cells grown on glass coverlips were stained with the FragEL DNA Fragmentation Kit for TUNEL analysis (Calbiochem; San Diego, CA), and counterstained with DAPI. TUNEL-positive cells and DAPI-stained nuclei were counted under a fluorescence microscope in ten 100× fields. The results are reported as percent apoptotic cells = (mean TUNEL-positive cells per field / mean total nuclei per field) × 100.

Embryoid bodies

Wild-type mouse embryonic stem (ES) cells and embryonic mouse fibroblast cells (EMFI) established as described (Su et al., 1999) were provided to us by Dr. A. Joyner (Memorial Sloan-Kettering Institute, New York). ES cells genetically deficient in either VEGF or VEGFR-1 or -2 were provided to us by Drs. Andras Nagy (Samuel Lunenfeld Research Institute, Toronto, Canada) (Carmeliet et al., 1996), Janet Rossant (University of Toronto, Ontario, Canada) (Fong et al., 1995) and Guo-Hua Fong (University of Connecticut) (Fong et al., 1995), respectively. ES cell culture and differentiation into embryoid bodies were performed as described (Gualandris et al., 2000a).

Western blotting

Western blotting was performed as described (Ferrari et al., 2006).

SiRna transfection

Subconfluent HUVE cells were incubated with 200 pmol of human VEGFR2 or VEGFR1 siRNA oligonucleotides (Dharmacon RNA technologies, Lafayette, CO) and 4 µl of Oligofectamine (Invitrogen, Carlsbad, CA) in serum-free medium for 4 h at 37° C, after which medium supplemented with 10% serum was added (Ferrari et al., 2006). The cells were used 48 h after transfection.