title: Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.
abstract: Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism.
authors.affiliation: From the Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6303.
authors.forename: William H
authors.identifier: http://orcid.org/0000-0003-3426-3709
authors.initials: WH
authors.lastname: Parker
authors.affiliation: From the Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6303.
authors.forename: Zhi-chao
authors.initials: ZC
authors.lastname: Qu
authors.affiliation: From the Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6303 james.may@vanderbilt.edu.
authors.forename: James M
authors.initials: JM
authors.lastname: May
mesh_terms: D000199:Actins / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology*; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007365:Intercellular Junctions / Q000187:drug effects / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism*; D008954:Models, Biological; D018994:Myosin Light Chains / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D058105:Polymerization / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000099152:Cadherin 5
chemical_list: D000199:Actins; D015703:Antigens, CD; D000975:Antioxidants; D001205:Ascorbic Acid; D015820:Cadherins; D002118:Calcium; D000242:Cyclic AMP; D006152:Cyclic GMP; D018994:Myosin Light Chains; D009569:Nitric Oxide; D013917:Thrombin; D020830:rac1 GTP-Binding Protein; D020827:rap1 GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D000099152:Cadherin 5
keywords: Ras-related C3 botulinum toxin substrate 1 (Rac1); Ras-related protein 1 (Rap1); actin; ascorbate; cyclic AMP (cAMP); cyclic GMP (cGMP); endothelial permeability; nitric oxide; paracellular transport; thrombin