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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 thrombininduced barrier permeabilization in human umbilical vein endothelial cells (HUVEC) 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 Rap1and 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. While 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. In vascular beds lacking significant fenestrations, the endothelium is the most significant barrier to the transfer of large serum proteins and other molecules from blood to the interstitium. Adherens junctions between cells contribute significantly to this barrier function by providing intercellular adhesion (1). These transmembrane protein junctions are stabilized by their intracellular association with the cortical actin cytoskeleton. During inflammation, the permeability of this barrier is increased due to formation of gaps between endothelial cells. These gaps are caused by the opening of adherens junctions and the centripetal tightening of the actin http://www.jbc. org/cgi/doi/10.1074/jbc.M115.662098 The latest version is at JBC Papers in Press. Published on July 7, 2015 as Manuscript M115.662098 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on M arch 4, 2020 hp://w w w . jb.org/ D ow nladed from Ascorbate prevents endothelial permeabilization by thrombin 2 cytoskeleton, which pulls the cells apart (2,3). It follows that many inflammatory mediators including thrombin, histamine and various oxidants compromise barrier integrity by diminishing cortical actin and promoting cytoskeletal rearrangement to form contractile actin stress fibers (2,4). In contrast, barrierstabilizing mediators such as cAMP, nitric oxide (NO) (5) and the small GTPases Rap1 and Rac1 (2) enhance adherens junctions and preserve