Figure 3.

Active Rac1 improves formation of vascular cords, organizes actin filaments cortically, and improves EC-EC junctions in vitro. (A) Dermal MVECs, transduced with Rac1 mutants or empty vector control, were induced to undergo capillary morphogenesis (cord formation, see “Methods”) by sandwiching between 2 layers of collagen I (top) or overlaying with collagen I (bottom). Subsequently cells were stained for F-actin. Top panels: note abundance of cord blind ends in DN Rac1 and control specimens that are absent in the active Rac1 specimen. Bottom panels (higher magnification): note cortical distribution of actin filaments in MVECs transduced with active Rac1, whereas actin is poorly organized in MVECs transduced with empty vector control or DN Rac1. (B) Quantification of cord parameters; n > 20 for all groups. Relative to controls, active Rac1 strongly reduced cord blind ends (P < .001) and increased cord integration as measured by counting closed polygons (P < .01), whereas DN Rac1 increased cord blind ends and reduced cord integration (polygons). (C) Confluent monolayers of MVECs, cultured in the presence of 20 ng/mL VEGF, and stained for F-actin (top panels) or VE-cadherin (bottom). Relative to empty vector control, active Rac1 strongly organized actin filaments cortically and improved integrity of EC-EC junctions as shown by VE-cadherin staining. DN Rac1 had opposite effects.