Tie-2 Knock-Down Inhibits MLC-p and Promotes Disrupted Endothelial Architecture, Replicating the Effects of Ang-2

<div><p>(A) HMVECs were stimulated with Ang-2 (100 ng/ml) in 2.5% FBS EBM-2 for the indicated times, and phospho-Tie-2 was detected by immunoprecipitation and Western blot (upper) as described in Methods. Similar amounts of total Tie-2 were present in HMVECs harvested at each time point. Phospho-Tie-2 declined over time whereas total Tie-2 remained relatively constant (lower).</p> <p>(B) Negative control siRNA (left column) or a Tie-2-specific siRNA (right column) was transfected in HMVECs. Cells were then serum-starved for 24 h, after which decreased Tie-2 expression (right column, upper blot) and increased MLC phosphorylation (right column, middle blot) were verified with Tie-2 siRNA.</p> <p>(C) Phase contrast (200×) and fluorescence images (600×) of cells stained for F-actin and VE-cadherin after transfection of negative control siRNA (panels a–d) or Tie-2 specific siRNA (panels e–h). Tie-2-siRNA caused thick actin stress fibers and gap formation in HMVECs (panel h, arrows). Phase contrast images, panels a and e; F-actin, panels b and f; VE-cadherin, panels c and g; merge images, panels d and h.</p>