siRNAs directed to MT1-MMP block EC lumen formation and generation of vascular guidance tunnels in 3D collagen matrices. (A) The graph shows the average corresponding lumen area in micrometers²  per high-powered field for each siRNA treatment. Cultures were examined at either 24 or 48 hours of culture. The inability of MT1-MMP siRNA treated cells to form lumens is shown, with the bars representing the average lumenal area ± SD (P < .01; n = 3). (B) The graph shows the average corresponding vascular guidance tunnel area in micrometers²  per high-powered field for each siRNA treatment. Individual fields were photographed under fluorescence after immunostaining of gels (24-hour cultures) with anti-collagen type I antibodies. Vascular guidance tunnel areas were traced using Metamorph software from the photographs. The inability of MT1-MMP siRNA-treated cells to form tunnels is shown, with the bars representing average tunnel area per field ± SD (P < .01). (C) Western blots showing MT1-MMP expression demonstrate specific knockdown of the gene with siRNA directed to MT1-MMP compared with MT3-MMP and MMP-1 as well as the control siRNA directed to luciferase. Actin was used as a loading control. (D) Representative images of siRNA-transfected ECs seeded within FITC-labeled collagen type I 3D matrices are shown. Luciferase (Luc), MT1-MMP, MT3-MMP, and MMP-1 siRNA-transfected mRFP-ECs were allowed to form lumens and tube networks for 48 hours and data quantified (A). Bar equals 100 μm.