Inhibitors of EC lumen formation block vascular guidance tunnel formation, and differential role of integrins in controlling EC motility and adhesive interactions with vascular guidance tunnel matrices. ECs were seeded into collagen matrices, and various inhibitors of lumen formation were added from the beginning of the assay as indicated. The indicated drugs (Go6976, Go6983, PP2) were each added at 10 μM, the integrin blocking antibodies were added at 20 μg/mL, and the MMP inhibitors, GM6001, and the recombinant TIMPs were added at 5 μM and 5 μg/mL, respectively. (A) Average luminal area was measured in micrometers2 from 5 independent cultures, with bars representing area ± SD (P < .01) and quantitated from images obtained from stained cultures. (B) Cultures were immunostained for collagen type I and quantification of average vascular guidance tunnel formation measured in micrometers2 from 5 independent cultures, with bars representing tunnel area ± SD (P < .01). (C) Nuclear GFP-EC cultures were established for 48 hours, after which time integrin blocking antibodies were added at 20 μg/mL and real-time imaging was performed to assess EC motility within vascular guidance tunnels. Velocity of migration was quantitated from 20 independent cell motility tracings from triplicate cultures and is calculated as μm per minute. (D) Schematic diagram showing that EC morphogenic processes lead to both lumen formation and vascular guidance tunnel formation. The lumen formation mechanism depends on MT1-MMP-dependent proteolysis and the α2β1 integrin in a 3D matrix environment. ECs initially are completely surrounded by collagen matrix. Vascular guidance tunnels which form as a consequence of EC lumen formation are then used as 2D migratory matrix surfaces allowing EC motility and tube remodeling events that are MT1-MMP-independent. The ECs flatten out within these tunnel spaces and are interacting with collagen ECM on their abluminal surfaces while their luminal surfaces are exposed to fluid, thus, mimicking a 2D matrix environment. EC migratory events involve αv and α2β1 integrins that recognize the proteolytically altered vascular guidance tunnel matrix surface (containing both native and denatured collagen type I).
