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

Journal:

Article Title: Up-regulating Sphingosine 1-Phosphate Receptor-2 Signaling Impairs Chemotactic, Wound-healing, and Morphogenetic Responses in Senescent Endothelial Cells * S⃞

doi: 10.1074/jbc.M804392200

Figure Lengend Snippet: S1P-mediated endothelial functions are impaired in senescent ECs. A, proliferative capabilities of young (Y, CPDL = 15), intermediate (I, CPDL = 35), and senescent (S, CPDL = 60) ECs. Cells were labeled with BrdUrd reagent for 24 h. Subsequently, cells were immunostained with anti-BrdUrd following the manufacturer's instructions (BrdUrd staining kit, Zymed Laboratories Inc.). Mean ± S.E. represents percentage of BrdUrd incorporated nuclei from 3-5 microscopic fields of duplicate experiments. B, chemotactic responses were measured by a Transwell migration assay as described under “Experimental Procedures.” Note that S1P (100 nm)-induced chemotactic responses were inversely related to in vitro ages of cultured ECs. C, young, intermediate, and senescent ECs were plated into each well of 8W1E arrays and incubated at 37 °C to allow the transendothelial electrical resistance (TEER) to reach equilibrium. 24 h later, the cells attached to the microelectrodes were killed by applying 5 V of electrical current for 30 s (arrow), which resulted in an abrupt impedance drop. Subsequently, endothelial wound-healing responses in the presence or absence of 100 nm S1P were determined in real-time by measuring the recovery of TEER, an indicator of endothelial migration into the wounded area (29). D, morphogenesis of young and senescent ECs on matrigel (upper panel) and quantitative analysis of tubular length (lower panel) in the presence or absence of S1P (200 nm) were performed as described (12, 13). Scale bar, 318 μm.

Article Snippet: Briefly, 200 μl of cell suspension (5 × 10 5 cells per ml) was seeded to each well of either an 8W1E or 8W10E ECIS array (Applied BioPhysics), which was pre-equilibrated with 200 μl of medium at 37 °C for 30 min. Two days later, endothelial cells were washed and replaced with plain M199 medium supplemented with 0.5% fetal bovine serum.

Techniques: Labeling, Staining, Transwell Migration Assay, In Vitro, Cell Culture, Incubation, Migration