| Loss of endothelial barrier function occurs as a part of microvascular dysfunction in patients with sepsis. Pathogenic bacteria, bacterial products such as endotoxin [e.g., Escherichia coli lipopolysaccharide (LPS)], and inflammatory cytokines [e.g. interferon-gamma (IFN-gamma)] trigger activation of intracellular signaling pathways in endothelial cells which contribute to endothelial barrier dysfunction. This dysfunction in microvessels endothelial layer is marked by an increased permeability to macromolecules, such as albumin, and frequently leads to plasma extravasation, edema, and organ failure. The purpose of this study was (i) to determine the mechanism by which the oxidized form of vitamin C, dehydroascorbate (DHA), may increase intracellular ascorbate during sepsis, and (ii) to determine the effectiveness of DHA and reduced form of vitamin C (ascorbate, AA) in preventing sepsis-induced microvascular dysfunction. In in-vitro experiments, LPS and IFN-gamma were used to induce septic insult in endothelial cells. The cells were then incubated with DHA to study DHA recycling, a process by which DHA is transported into endothelial cells and rapidly reduced to AA. For in-vivo experiments, sepsis was induced in mice by laprotomy and cecal ligation and puncture (CLP) procedures. Later, Evans-blue dye was injected into the jugular vein to test endothelial barrier dysfunction. HPLC based electrochemical detection assay was used to determine intracellular concentration of AA. Protein expression was detected by western blot technique and endothelial permeability was measured on the basis of Evans-blue dye extravasation. Results from in-vitro studies revealed that DHA recycling was accelerated in endothelial cells stimulated with LPS and IFN-gamma. The data also showed that LPS and IFN-gamma induced DHA recycling depended on NADPH oxidase. Results from in-vivo studies showed that mice that underwent CLP developed microvascular dysfunction in the heart and lungs. Administration of AA and DHA intravenously, prior to sepsis-inducing CLP procedure, decreased microvascular dysfunction in the heart. AA and DHA were equally effective. Taken together, these results support the conclusion that administration of DHA or AA is beneficial for preventing septic microvascular dysfunction. |
