Fig. 3

Effect of UFH pretreatment on LPS-induced increase in endothelial cell permeability and F-actin remodeling. HPMECs were pretreated with vehicle or UFH, followed by LPS stimulation for 6 h. a The TEER across the cells were measured. UFH (≥ 10 U/ml) significantly protected cells from LPS-stimulated endothelial barrier dysfunction. b The influx of FITC-conjugated dextran across cells was determined. UFH (10 U/ml) pretreatment decreased LPS-stimulated hyperpermeability. c Actin cytoskeletal remodeling and area of inter-endothelial gaps. Actin cytoskeletal remodeling was examined by immunofluorescence staining with TRITC-phalloidin. Arrows indicate intercellular gaps. Vehicle and UFH group displayed intact monolayer, and stress fibers were rarely observed. UFH pretreatment decreased LPS-induced stress fibers and intercellular gaps (scale bars = 50 μm). The area of the gaps in the microscope fields was assessed and normalized to the area of gaps induced with LPS alone treatment. UFH pretreatment prevented the LPS-induced changes in the actin cytoskeleton. For each group, nine microscope fields from three parallel experiments were analyzed. d Western blot analysis was used to evaluate GEF-H1 levels and MYPT1 phosphorylation. Equal protein loading was confirmed by GAPDH levels. Data are representative of three independent experiments. t-test, * P < 0.05 compared to the vehicle group; t-test, # P < 0.05 compared to the LPS-treated group