Ang-1 elevates Rac1 activity in MVECs and exerts Rac1-dependent improvement of cord formation, cortical actin, and EC-EC junctions. (A) Ang-1 (100 ng/mL, 24 hours) elevates Rac1 activity in dermal MVECs. (B) Ang-1 (100 ng/mL) improves cord formation by MVECs sandwiched between 2 layers of collagen I (see “Methods”). Cells were stained for F-actin. Note abundance of cord blind ends in vehicle control and Ang-1 + Rac1 inhibitor (NSC23766 50μM, ∼ 1× median effective concentration) specimens that are absent in the Ang-1 specimen. (C) Quantification of cord parameters; n > 25 for all groups. Relative to controls, Ang-1 reduced cord blind ends (P < .01) and increased cord integration as measured by counting closed polygons (P < .01). Rac1 inhibitor NSC23766 abolished these effects, indicating that Ang-1 improves cord formation through a Rac1-dependent mechanism. (D) Confluent monolayers of MVECs, cultured in the presence of 20 ng/mL VEGF, and stained for F-actin (top) or VE-cadherin (bottom). Relative to empty vector control, Ang-1 (100 ng/mL, 24 hours) strongly organized actin filaments cortically and improved integrity of EC-EC junctions as shown by VE-cadherin staining. Within 1 hour, Rac1 inhibitor NSC23766 (50μM, ∼ 1× median effective concentration) abolished Ang-1–mediated enhancement of cortical actin and EC-EC junctions.