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Rnd3 inhibits thrombin‐induced endothelial barrier dysfunction
Author(s) -
Breslin Jerome W,
Daines Dayle A,
Wu Mack H,
Yuan Sarah Y
Publication year - 2008
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.22.1_supplement.926.6
Subject(s) - microbiology and biotechnology , thrombin , umbilical vein , chemistry , blot , endothelial stem cell , phosphorylation , immunoprecipitation , endothelium , barrier function , platelet , biology , biochemistry , immunology , in vitro , endocrinology , gene
The small GTPase Rho has previously been shown to be involved in elevated microvascular permeability. Recently, a new member of the Rho family, Rnd3, was described as an endogenous inhibitor of Rho signaling. We hypothesized that Rnd3 promotes endothelial barrier integrity. To test this hypothesis, we evaluated barrier function of cultured human umbilical vein endothelial cells (HUVEC) under basal conditions and after stimulation with 1 U/ml thrombin. To test the role of Rnd3, we either overexpressed a MAT‐FLAG‐Rnd3 fusion protein or selectively knocked down expression of endogenous Rnd3 with siRNA. Expression levels were confirmed by Western blotting. Endothelial barrier function was evaluated by measuring transendothelial electrical resistance or by determining the permeability to FITC‐albumin in cultured HUVEC monolayers. We also evaluated whether thrombin alters Rnd3 localization using immunofluorescence microscopy, as well as Ser and Tyr phosphorylation of Rnd3, with immunoprecipitation and Western blotting. The results show that overexpression of Rnd3 in HUVEC attenuates thrombin‐induced endothelial hyperpermeability, whereas selective knockdown of Rnd3 extended the time‐course. In addition, we observed that Rnd3 mainly localizes in the perinuclear region of unstimulated endothelial cells but distributed throughout the cytoplasm and in some cases at the plasma membrane in thrombin‐treated cells. Thrombin also increased the degree of Ser and Tyr phosphorylation of Rnd3. These findings suggest that Rnd3 promotes endothelial barrier integrity, and that thrombin may affect endothelial permeability via posttranslational modification of Rnd3 in endothelial cells. Supported by NIH grants HL61507, HL70752, and HL73324.