Pericyte TIMP-3 regulates basement membrane formation during EC-pericyte coassembly events within vascular guidance tunnels during tube remodeling and maturation events. (A) Immunostaining of basement membrane components reveals that in cocultures in which pericytes were treated with a siRNA to TIMP-3, there is marked disruption of collagen IV (first column in red, with the second column showing overlays denoting the position of GFP-pericytes). (B) Furthermore, suppression of pericyte TIMP-3 leads to increased vessel width (P < .01). (C) RT-PCR analysis of pericyte TIMP-3 suggests regulation over time in EC-pericyte cocultures. G3PDH is shown as a loading control. (D) Western blot analysis of TIMP-3 is shown to demonstrate siRNA suppression versus controls. (E) This schematic diagram illustrates the function of vascular guidance tunnels²⁴  that affect EC tube remodeling, recruitment of pericytes, and dynamic EC-pericyte interactions that are necessary for deposition of basement membrane matrix as well as EC and pericyte integrin expression changes that control vascular tube maturation events. Vascular guidance tunnels form as a consequence of EC lumen and tube formation that occurs through a signaling cascade involving Cdc42, MT1-MMP, and α₂β₁ integrin and the indicated downstream kinase effectors.^35,36  Asterisks indicate basement membrane components and integrins that are up-regulated specifically by EC-pericyte interactions during tube coassembly in 3D collagen matrices. Cross indicates the down-regulation of α₂β₁, which is observed in EC-pericyte cocultures and not EC-only cultures.