MMP activity is required for EC lumen formation and generation of vascular guidance tunnels in 3D collagen matrices. (A) EC cultures were established with or without the addition of the proteinase inhibitors GM6001 (5 μM; top panels) or recombinant TIMP-3 (5 μg/mL; bottom panels). Collagen gels were fixed at 24 hours and processed for immunostaining of the collagen type I matrix (top panels) or were fixed after 48 hours (bottom panels). (Top panels) White arrows indicate the outline of vascular guidance tunnels. Bar equals 50 μm. (Bottom panels) Representative light microscopy images are shown demonstrating quantification of EC lumen formation with and without TIMP-3 addition. Bar equals 100 μm. (B) GFP-ECs were seeded within collagen matrices and allowed to form lumens and tube networks. Cultures were fixed at 96 hours and immunostained for the collagen type I matrix using a collagen type I monoclonal antibody and an Alexa Fluor 594 conjugated secondary antibody. Representative fluorescent images are shown which illustrate that ECs undergo morphogenesis within vascular guidance tunnels. Arrows denote the borders of vascular guidance tunnels. 10×, bar equals 100 μm; 40×, bar equals 25 μm. (C) ECs were suspended in 3D collagen gels and allowed to undergo morphogenesis for 48 hours. Lumen areas per field were determined by tracing EC lumens using Metamorph software from time-lapse images at the indicated time points. The effects of exogenous addition of TIMPs 1-4 and GM6001 on EC lumen formation over the time course is shown, with the adjacent bar graph highlighting EC lumen area at the final 48-hour time point. n = 3 fields per time point.