Rat Aorta Angiogenesis Bioassay

The rat aorta angiogenesis bioassay [33 (link)] was modified to enhance the ability to screen for pro-angiogenic activity in the HPLC-derived fractions by reducing the concentration of heat inactivated foetal calf serum (HIFCS) from 20% to 5% and incubating for 7 days in 48 well culture plates (Costar, Corning, Lowell, MA) at 37°C in 5% CO₂ [36 (link)]. Six technical and three biological replicates were used and the medium was changed on day 4 of culture. Vessel outgrowth was quantified as percent growth under a Nikon TMS inverted phase contrast microscope (Nikon Instrument Inc., Tokyo, Japan) at 40 × magnification on days 5, 6 and 7. Control cultures either received medium with the diluent alone or with the known anti-angiogenic agent, PI-88 [33 (link)] added at 100 μg/mL. Vessel outgrowth was determined as the percentage of a microscope field, outside the aorta ring, that was occupied by blood vessels using ImageJ software as previously described [40 (link)] or measured manually, particularly when large numbers of column fractions needed to be scored.