CGMP and VASP in Oxidant Endothelial Dysfunction

Johns Hopkins University, Baltimore MD

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Abstract

[unreadable] DESCRIPTION (provided by applicant): Acute lung injury often results from endothelial cell (EC) barrier dysfunction secondary to reactive oxygen species (ROS). Treatments that increase cGMP decrease this EC dysfunction by an unknown mechanism. Our objective is to examine the molecular mechanisms by which cGMP prevents ROS-induced EC barrier dysfunction. In EC, cGMP activates cyclic G kinase (cGK), which phosphorylates vasodilator-stimulated phosphoprotein (VASP). Unphosphorylated VASP binds to cytoskeletal F-actin and proteins in cell-cell and cell-matrix attachments and promotes F-actin stress fiber formation and cell ruffling; the effect on EC permeability is unknown Phosphorylation of VASP reverses these effects. Our preliminary data in pulmonary conduit and microvascular EC monolayers show that H2O2 decreases cGMP and barrier function with cytoskeletal rearrangement and increased [Ca2+]i cGMP prevents the H2O2-induced changes and enhances baseline EC barrier function. We hypothesize that CBMP prevents a H2O2-induced decrease in the P-VASP/VASP ratio by 1) increasing P-VASP through cGK1a and 2) decreasing phosphatase (PPase) activity, in part through decreased [Ca2+]i. In Aim 1, assessments of permeability and cytoskeletal structure following cGK1a agonists/antagonists and over-expression of dominant negative and constitutively active cGK1a constructs, will define the role of cGK1a in H2O2 induced events. Aim 2 will examine VASP phosphorylation as the key molecular switch mediating the opposing effects of cGMP and H2O2. Measurements of barrier function following transfection with antisense and phosphorylation-resistant VASP will define its function and highlight critical phosphorylation sites. In Aim 3, the VASP PPase will be identified using specific inhibitors, activity assays, and co-localization experiments. PPase regulation by CGK1a [Ca2+]i, and H2O2 will be determined and the effect of over-expression vs. inhibition of PPase on VASP and H2O2 included barrier dysfunction will be examined. A better understanding of cGMP-induced EC barrier protection will provide a basis for new treatments of ROS-mediated vascular disease.