Figure 3

Stress fiber assembly in HIMEC following VEGF stimulation. VEGF (10 ng/ml) induced stress fiber assembly was attenuated by anti-VEGFR2, CsA and SB203580 but not PD098059. The effect of VEGF on stress fiber polymerization in HIMEC was assessed by fluorescence staining with fluorescein phalloidin, a substance which specifically detects F-actin. Confluent HIMEC monolayers were grown on fibronectin-coated glass chamber slides, and stimulated with VEGF (1–10 ng/ml, 15 min) prior to staining. Control, minimal stress fibers are demonstrated in unstimulated HIMEC (negative control). VEGF, at 10 ng/ml but not at 50 ng/ml strongly increased stress fiber assembly. At some intercellular junctions, a marked retraction of endothelial cells is notable, leading to scattered interruptions in continuity of the endothelial cell monolayer. Pre-incubation of HIMEC with SB203580 (p38 MAPK inhibitor, 5 μM), CsA (0.1 μM) and neutralizing anti-VEGFR2 antibody (10 μg/ml, 15 min, 37°C) markedly attenuated stress fiber assembly by VEGF (note diminished fluorescence intensity and less pronounced stress fiber induction). Fluorescence microscopic images were obtained with an original magnification of 400×, at a fixed shutter speed. Figure shows representative images from one of three independent experiments.