Journal: Journal of Intensive Care

Article Title: Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients

doi: 10.1186/s40560-018-0289-5

Figure Lengend Snippet: Endothelial barrier disruption by neutrophils isolated from healthy ( n = 14), trauma ( n = 8), and septic donors ( n = 6) and protection by LA-1. Change in normalized resistance with respect to time. T = 0 represents time of neutrophil addition to HUVECs activated by TNF for 4 h. a Representative tracing of electrical resistance across activated HUVEC monolayers in the presence of neutrophils obtained from healthy, trauma, or septic donors. b Values are mean ± SEM of normalized resistance at 60 min following PMN addition. Analyses were performed evaluating the effect of neutrophils obtained from critically ill trauma patients ( n = 8), from critically ill septic patients ( n = 6), and from healthy controls ( n = 14) and assayed in duplicate on each experimental day. c Representative ECIS tracing of neutrophils added to HUVEC monolayer 30 min after fMLP (10 −6 M). d Values are mean ± SEM of normalized resistance at 60 min following PMN addition ± fMLP. e – g Representative tracings of neutrophils ± fMLP activation in the presence or absence of LA-1. e Neutrophils obtained from healthy donors. f Neutrophils obtained from trauma patients. g Neutrophils obtained from septic patients. h Values are mean ± SEM of normalized resistance at 60 min following PMN addition ± fMLP. * p < 0.05 vs. healthy unstimulated; ** p < 0.05 vs. healthy fMLP-stimulated; # p < 0.05 vs. trauma unstimulated; ## p < 0.05 vs. trauma fMLP-stimulated; @ p < 0.05 vs. septic unstimulated; and @@ p < 0.05 vs. septic fMLP-stimulated. Statistical significance determined ( p < 0.05) using three-way ANOVA with post hoc Tukey analysis

Article Snippet: Electric cell-substrate impedance sensing (ECIS) cultureware electrode arrays (8W10E+) and 16-well array station were obtained from Applied BioPhysics (Troy, NY).

Techniques: Disruption, Isolation, Activation Assay

Journal: Scientific Reports

Article Title: A CDC42-centered signaling unit is a dominant positive regulator of endothelial integrity

doi: 10.1038/s41598-017-10392-0

Figure Lengend Snippet: Functional RhoGTPases siRNA screen identifies new regulators of the endothelial barrier. ( a ) Schematic representation of the siRNA screen. Upper left corner, clockwise: the selected 270 siRNA targets distributed by functional group: RhoGTPases (22), RhoGEFs (82), RhoGAPs (66), RhoGDIs (3), Rho-effector proteins (76) and Rho-associated genes (21). 270 SMARTpools and control siRNAs were forward transfected into primary HUVECs seeded on 96W10idf ECIS arrays (n = 3 independent experiments). Endothelial barrier resistance was measured for 72 h following transfection by ECIS. Typical graph of the ECIS readout is shown. Depicted in green are exemplary readings of genes that enhance barrier function after knock-down; in red, genes that decrease barrier function upon loss-of-function; in black, a control non-targeting siRNA. Statistical analysis was performed and hits were identified as detailed in Results. Validation experiments were performed on the top hits. ( b ) Overview of individual mean endothelial resistance values registered at 72 h post-transfection of each siRNA pool (black dots) (n = 3). The average resistance of the non-targeting siRNA controls is represented by the background blue line. (n = 54). In grey, the threshold value set at ±200 ohm on the average non-targeting siRNA controls. Statistically significant hits, not all of which also induced large deviations in barrier function, are indicated by circles. ( c ) Distribution of 10 top hits by functional group.

Article Snippet: Briefly, passage 2 cells (1 × 10 4 cells/well) were seeded on 1% gelatin-coated 96w10idf or (1 × 10 5 cells/well) 8w10E arrays connected to the ECIS®ZTheta Array Station (Applied Biophysics, Troy, NY) and transfected as described above.

Techniques: Functional Assay, Transfection