S A Schlidt | PubMed Health

PubMed1999

Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction.

Prolonged hypoxia produces reversible changes in endothelial permeability, but the mechanisms involved are not fully known. Previous studies have implicated reactive oxygen species (ROS) and cytokines in the regulation of permeability. We tested whether prolonged hypoxia alters permeability to increasing ROS generation, which amplifies cytokine production. Human umbilical vein endothelial cell (HUVEC) monolayers were exposed to hypoxia while secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-6, and IL-8 was measured. IL-6 and IL-8 secretion increased fourfold over 24 h in a pattern corresponding to changes in HUVEC permeability measured by transendothelial electrical resistance (TEER). Addition of exogenous IL-6 to normoxic HUVEC monolayers caused time-dependent changes in TEER that mimicked the hypoxic response. An antibody to IL-6 significantly attenuated the hypoxia-induced changes in TEER (86 +/- 4 vs. 63 +/- 3% with hypoxia alone at 18 h), whereas treatment with anti-IL-8 had no effect. To determine the role of hypoxia-induced ROS on this response, HUVEC monolayers were incubated with the antioxidants ebselen (50 microM) and N-acetyl-L-cysteine (NAC, 1 mM) before hypoxia. Antioxidants attenuated hypoxia-induced IL-6 secretion (13 +/- 2 pg/ml with ebselen and 19 +/- 3 pg/ml with NAC vs. 140 +/- 15 pg/ml with hypoxia). Ebselen and NAC prevented changes in TEER during hypoxia (94 +/- 2% with ebselen and 90 +/- 6% with NAC vs. 63 +/- 3% with hypoxia at 18 h). N-nitro-L-arginine (500 microM) did not decrease hypoxia-induced changes in dichlorofluorescin fluorescence, IL-6 secretion, or TEER. Thus ROS generated during hypoxia act as signaling elements, regulating secretion of the proinflammatory cytokines that lead to alterations of endothelial permeability.

Antibodies↗

PubMed1998

Prolonged hypoxia alters endothelial barrier function.

BACKGROUND: It is well recognized that hypoxia/reoxygenation and exposure to inflammatory mediators such as cytokines and neutrophils alter the barrier function of the vascular endothelium. The experiments we conducted tested whether hypoxia alone could produce changes in permeability and whether a prolonged period of hypoxia alters the surface expression of cell adhesion molecules. METHODS: Endothelial cells were cultured from human umbilical vein endothelial cells (HUVECs). Hypoxia was created by isolating the cells in a chamber through which 1% 02, 5% CO2, and 94% N2 were insufflated (30 min at 1/min). Oxygen tension was measured through oxygen-quenching phosphorescence. Hypoxia was maintained for 24 hours. Changes in endothelial permeability were measured by transendothelial electrical resistance (TEER). Endothelial leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression were assessed by flow cytometry (mean +/ standard error of the mean [SEM]. RESULTS: Exposure of endothelial cells to hypoxia resulted in increased permeability between 6 and 24 hours, with the greatest decrease in TEER at 18 hours (63% +/ 3%; P < .05). Prolonged hypoxia produced no change in the surface expression of ELAM-1 or ICAM-1. CONCLUSIONS: Hypoxia alone produced a significant reversible alteration in endothelial permeability. However, this change was observed only under severe hypoxic conditions (eg, below 20 mm Hg); higher oxygen tensions (25 and 35 mm Hg) had no significant effect. Unlike observations made after cytokine exposure, hypoxic breakdown of endothelial barrier function was unassociated with up-regulation of either ELAM-1 or ICAM-1.

Cell Hypoxia↗