Abstract
Regulation of actin dynamics is critical for endothelial barrier functions. We provide evidence that the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) is required for endothelial barrier maintenance. Baseline permeability was significantly increased in VASP-deficient (VASP(-/-)) microvascular myocardial endothelial cells (MyEnd) in the absence of discernible alterations of immunostaining for adherens and tight junctions. We tested whether VASP is involved in the endothelium-stabilizing effects of cAMP or Rac 1. Forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 (CNF-1) to activate Rac 1 were equally efficient to stabilize barrier functions in VASP(-/-) and wild-type (wt) cells. In wt cells, VASP was phosphorylated in response to F/R but did not localize to intercellular junctions. In contrast, CNF-1 and expression of constitutively active Rac 1 induced translocation of VASP to cell borders in wt cells, where it colocalized with active Rac 1. In VASP(-/-) cells, Rac 1 activity was reduced to 0.4 of wt levels in controls and increased approximately 20-fold in response to CNF-1 compared with 7-fold activation in wt cells. Moreover, inactivation of Rac 1 by lethal toxin led to a greater increase of permeability compared with wt cells. All these data suggest that VASP is involved in the regulation of Rac 1 activity. Taking these findings together, our study indicates that VASP at least in part stabilizes endothelial barrier functions by control of Rho-family GTPases.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenylyl Cyclases / metabolism
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Animals
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Antigens, CD / metabolism
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Bacterial Toxins / pharmacology
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Cadherins / metabolism
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Capillary Permeability* / drug effects
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Cell Adhesion
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Cell Adhesion Molecules / deficiency
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Cell Adhesion Molecules / genetics
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Cell Adhesion Molecules / metabolism*
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Cell Line
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Colforsin / pharmacology
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Coronary Vessels / drug effects
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Coronary Vessels / enzymology
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Coronary Vessels / metabolism*
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Cortactin / metabolism
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Cyclic AMP / metabolism*
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Endothelial Cells / drug effects
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Endothelial Cells / enzymology
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Endothelial Cells / metabolism*
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Enzyme Activators / pharmacology
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Escherichia coli Proteins / pharmacology
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Intercellular Junctions / metabolism
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Mice
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Mice, Transgenic
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Microfilament Proteins / deficiency
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Microfilament Proteins / genetics
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Microfilament Proteins / metabolism*
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Neuropeptides / genetics
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Neuropeptides / metabolism*
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Phosphodiesterase Inhibitors / pharmacology
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Phosphoproteins / deficiency
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Phosphoproteins / genetics
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Phosphoproteins / metabolism*
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Phosphorylation
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Protein Transport
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Rolipram / pharmacology
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Signal Transduction* / drug effects
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Transfection
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rac GTP-Binding Proteins / genetics
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rac GTP-Binding Proteins / metabolism*
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rac1 GTP-Binding Protein
Substances
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Antigens, CD
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Bacterial Toxins
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Cadherins
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Cell Adhesion Molecules
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Cortactin
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Cttn protein, mouse
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Enzyme Activators
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Escherichia coli Proteins
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Microfilament Proteins
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Neuropeptides
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Phosphodiesterase Inhibitors
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Phosphoproteins
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Rac1 protein, mouse
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cadherin 5
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vasodilator-stimulated phosphoprotein
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cytotoxic necrotizing factor type 1
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Colforsin
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Cyclic AMP
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rac GTP-Binding Proteins
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rac1 GTP-Binding Protein
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Adenylyl Cyclases
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Rolipram