Rationale Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of eNOS to cytosol and stimulation of eNOS-derived NO signaling cascade. The mechanisms by which NO signaling regulates permeability at adherens junctions are still incompletely understood. Objective We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. Methods and Results We measured PAF-stimulated S-nitrosation of β-catenin and p120-catenin (p120) in three cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein) and CVEC (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of β-catenin and p120 at the adherens junction and with hyperpermeability. TNF-α increased NO production and caused similar increase in S-nitrosation as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S-nitrosation of β-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of NO production and of S-nitrosation blocked PAF-induced S-nitrosation and hyperpermeability whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with VE-cadherin. Conclusions Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.
We tested the hypothesis that platelet-activating factor (PAF) induces -nitrosylation of vasodilator-stimulated phosphoprotein (VASP) as a mechanism to reduce microvascular endothelial barrier integrity and stimulate hyperpermeability. PAF elevated-nitrosylation of VASP above baseline levels in different endothelial cells and caused hyperpermeability. To ascertain the importance of endothelial nitric oxide synthase (eNOS) subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced -nitrosylation of VASP in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Reconstitution of VASP knockout myocardial endothelial cells with cysteine mutants of VASP demonstrated that-nitrosylation of cysteine 64 is associated with PAF-induced hyperpermeability. We propose that regulation of VASP contributes to endothelial cell barrier integrity and to the onset of hyperpermeability. -nitrosylation of VASP inhibits its function in barrier integrity and leads to endothelial monolayer hyperpermeability in response to PAF, a representative proinflammatory agonist. Here, we demonstrate that -nitrosylation of vasodilator-stimulated phosphoprotein (VASP) on C64 is a mechanism for the onset of platelet-activating factor-induced hyperpermeability. Our results reveal a dual role of VASP in endothelial permeability. In addition to its well-documented function in barrier integrity, we show that-nitrosylation of VASP contributes to the onset of endothelial permeability.
Platelet‐activating factor (PAF) increases endothelial permeability (hyperpermeability) via endothelial NOS (eNOS), which stimulates cGMP production to enhance permeability. We explored the complementary hypothesis that PAF‐stimulated hyperpermeability involves eNOS‐derived NO induced S‐nitrosylation of adherens junction proteins. We measured PAF‐stimulated S‐nitrosylation of β‐catenin and p120‐catenin (p120) in 3 cell lines: ECV‐eNOSGFP, EAhy926 and CVEC. S‐nitrosylation reduced expression of β‐catenin and p120 at the adherens junction and enhanced permeability. To ascertain the significance of eNOS location, we used ECV‐304 cells transfected with constructs that target eNOS to the cytosol (G2AeNOSGFP) and plasma membrane (CAAXeNOSGFP). We detected S‐nitrosylation of β‐catenin and p120 only in ECV‐GFPeNOS‐G2A, i.e., in cells with cytosolic eNOS. PAF‐stimulated S‐nitrosylation decreased the association between β‐catenin and p‐120. PAF significantly decreased the association between β‐catenin and p‐120 only in ECV‐GFPeNOS‐G2A. Inhibitors of NO production and of S‐nitrosylation blocked PAF‐induced S‐nitrosylation and hyperpermeability. Our results demonstrate that agonist‐induced S‐nitrosylation contributes to junctional membrane protein changes that enhance endothelial permeability.
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