Premium
Evidence for a Rac1 independent mechanism for sphingosine‐1‐phosphate mediated endothelial barrier enhancement (672.7)
Author(s) -
Zhang Xun,
Adderley Shaquria,
Breslin Jerome
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.672.7
Subject(s) - lamellipodium , transfection , rac1 , microbiology and biotechnology , gene knockdown , small interfering rna , small gtpase , chemistry , biology , cell migration , signal transduction , cell , biochemistry , apoptosis , gene
S1P is a bioactive lipid that tightens intercellular junctions between endothelial cells (EC) through coordinated spreading of local lamellipodia. We tested the hypothesis that S1P causes protrusions through activation of the small GTPase Rac1. Cultured human umbilical vein EC or human dermal microvascular EC monolayers were used to model the endothelial barrier. Trans‐endothelial electrical resistance (TER) served as an index of barrier function and time‐lapse imaging of EC was employed to study local lamellipodia. Rac1 expression/activity was modulated by transfection of siRNA or plasmids encoding wild‐type (WT) or dominant‐negative (DN) GFP‐Rac1, and confirmed by Western blot or fluorescence microscopy, respectively. Non‐targeting RNA sequences and GFP transfection served as respective controls. The results show that S1P (2 μM) caused rapid, coordinated protrusions of local lamellipodia from individual cells that coincided with increased TER. Neither Rac1 knockdown, nor overexpression with WT or DN Rac1 significantly changed TER, compared to controls, at 72h. Additionally, S1P significantly increased TER in Rac1 knockdown and overexpression groups, at an equivalent magnitude as controls. These data suggest that a Rac1 independent mechanism controls S1P‐induced EC protrusions and endothelial barrier enhancement. Supported by NIH R01HL098215. Grant Funding Source : Supported by NIH R01HL098215