Figure 5From: Plant proteolytic enzyme papain abrogates angiogenic activation of human umbilical vein endothelial cells (HUVEC) in vitro Effect of proteolytic enzymes on cell migration and tube formation in HUVEC. HUVEC were seeded into fibronectin coated 12-well microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM. Cells were stained using calcein-AM, the monolayer was scratched with a 200 μL pipette tip and photographed at 10 fold magnification using a Nikon Eclipse Ti as described above. After 14 hours culture in EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and papain and photographed. covered area was measured using the TScratch software package, results were calculated as percent open area. Data are shown as mean ± SEM. Panel A shows photomicrographs at the beginning of the assay (column start) and after 14 hours incubation (column end) for cells treated with either 10 ng/mL VEGF or 10 ng/mL VEGF and 10 μg/mL papain. Migration fronts are marked by white lines. Panel B shows the percentage open area. Papain inhibited cell migration almost completely at a concentration of 10 μg/mL. Angiogenesis slides were coated with 10 μL Matrigel per well and incubated for 30 min at 37°C. Endothelial cells were and seeded into the wells at a density of 5000 cells per well in EBM containing 10 ng/mL VEGF. After 4 hours preincubation, papain was added at concentrations as indicated. After a further 20 hour incubation period, cells were labelled with 2 μM Calcein-AM. Micrographs of fluorescent cells were taken at 4 fold magnification using a Nikon Eclipse Ti as described above. Tube formation was quantified by the angiogenesis analyzer plugin for ImageJ. Panel C shows photomicrographs for the control, treatment with 10 ng/mL VEGF and with 10 ng/mL VEGF in combination with 1 and 10 μg/mL papain. Panel D shows the measured network length as percentage untreated control. Tube length decreased significantly to control levels after treatment with 1 μg/mL papain. At concentrations of 10 μg/mL tube formation was almost completely abrogated.Back to article page