FIGURE 3.
EC migration and in vitro tube formation in response to VEGF requires uPA. A, migration of lung microvascular ECs isolated from WT or uPA−/− mice in response to VEGF. LMVECs were starved in EBM-2/0.5% FBS for 24 h, detached with trypsin/EDTA, washed in starvation medium, and resuspended in the same medium. Cell suspensions were added inside FluoroblokTM transwells, which were placed in 24-well plates containing either starvation medium or the same medium supplemented with 25 ng/ml VEGF. Transwells were incubated with calcein AM and Hoechst to visualize migrating cells and then fixed. Cells were allowed to migrate for 18 h and were then photographed with a 4× objective using the EVOS FL Auto Imaging System microscope. Cells within the imaged field were counted. Each condition was set up in three wells, and three images were taken at different sites within each transwell. Typical images for each condition are shown. The bar graph on the right shows the mean ± S.E. cell numbers per microscopic field of WT or uPA−/− cells that migrated in response to VEGF. Few cells migrated in starvation medium (two-three per microscopic field) and, therefore, do not appear on the graph. *, p < 0.001. B, tubular network formation by WT and uPA−/− ECs in response to VEGF. WT and uPA−/− ECs were allowed to adhere and migrate within a 24-well plate coated with Matrigel. The endothelial cell network was visualized by loading the cells with Calcein AM dye (4 μg/ml), and photographs were taken using an EVOS® FL Auto Imaging System microscope. Typical images for each condition are shown on the left. Total length of the tubes was enumerated using the ImageJ software. *, p < 0.001 (right).