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.