In vitro, S1P₁ agonist SEW2871 exerts Rac-dependent improvement of vascular cords, cortical actin, and EC-EC junctions. (A) Dermal MVECs, treated with SEW2871 (15nM, ∼ 1× median effective concentration), vehicle, or SEW2871 + Rac1 inhibitor NSC23766 (50μM, ∼ 1× IC₅₀) were induced to form capillary cords by sandwiching between 2 layers of collagen I (see “Methods”). Subsequently cells were stained for F-actin. Note abundance of cord blind ends in vehicle control and SEW2871 + Rac inhibitor specimens that are absent in the SEW2871 specimen. (B) Quantification of cord parameters; n > 25 for all groups. Relative to controls, SEW2871 strongly reduced cord blind ends (P < .001) and increased cord integration as measured by counting closed polygons (P < .001). Rac1 inhibitor NSC23766 abolished these improvements, indicating that SEW2871-mediated enhancement of cord formation is Rac1 dependent. (C) 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, SEW2871 (15nM, ∼ 1× median effective concentration, 24 hours) strongly organized actin filaments cortically and improved integrity of EC-EC junctions as highlighted by VE-cadherin staining. Addition of Rac1 inhibitor NSC23766 (50μM, ∼ 1× IC₅₀, 1 hour) abolished SEW2871-mediated enhancement of cortical actin and EC-EC junctions.