Fig. 1

Endotoxin induces endothelial hyperpermeability through a mechanism mediated by TLR4/NOX/ROS/NF-κB pathway. a Endotoxin increases EC monolayer permeability. ECs were exposed to endotoxin (0, 10, 20, 35, and 50 μg/ml) for 24 h and endothelial permeability to FITC-dextran was analyzed. (N = 8). b Endotoxin induces a decrease in VE-cadherin expression. ECs were exposed to endotoxin (0, 10, 20, 35, and 50 μg/ml) for 24 h and VE-cadherin protein expression was analyzed. Protein levels were normalized against tubulin. Data are expressed relative to control (N = 5). c–k Endotoxin induces endothelial hyperpermeability through the TLR4/NOX/ROS/NF-κB pathway. ECs were preincubated for 1 h in the presence or absence of TLR4 inhibitor CLI-095 (c), NOX inhibitors DPI (d) and apocynin (e), antioxidant agent NAC (i), reducing molecule GSH (j), or NF-κB inhibitor SC3060 (preincubated for 6 h) (k). A different set of ECs were transfected using siRNAs against NOX isoforms NOX-1 (f), NOX-2 (g), and NOX-4 (h). Cells were then exposed to endotoxin (20 μg/ml) for 24 h, after which, endothelial permeability of FITC-dextran was analyzed. (N = 4–9). Results are expressed as normalized permeability compared with control. Statistical differences were assessed by one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 against control condition. Histogram bars show mean ± SD