Mechanism of inhibitory effect of hypoxia/reoxygenation on gap junctional intercellular communication of vascular endothelial cells in culture

We studied the change in gap junctional intercellular communication (GJIC) on human umbilical vein endothelial cells (HUVECs) under hypoxia/reoxygenation (H/R) conditions and the influence of leukocytes on GJIC by the fluorescence redistribution after photobleaching (FRAP) method. Contrast microscopic observations showed no significant changes in the morphology of the HUVECs after H/R. Reoxygenation following hypoxia (12 h) caused a time-dependent decrease in GJIC; that is, GJIC reduction was induced after 2 h and reached maximum levels at 4-6 h, recovering to normal levels after 18 h. The oxidant-sensitive fluorescence dye assay revealed that generation of reactive oxygen species (ROS) increased during the first 2 h after reoxygenation. Hydroxyl radical scavengers abolished reduction of GJIC by H/R. H/R led to significant release of membrane and supernatant tumor necrosis factor-alpha (TNF alpha). Anti-human TNF alpha antibody abolished the H/R-induced suppression of GJIC. GJIC of HUVECs that had adherent neutrophils was suppressed. Treatment with anti-human intercellular adhesion molecule-1 (ICAM-1) antibody abolished this suppression. Assays using intercell chambers showed that no suppression of GJIC was observed. This suppression was abolished by pretreatment with tyrosine kinase inhibitors. These data suggest that (1) ischemia/reperfusion injury may be involved in suppression of GJIC induced by TNF alpha release via reactive oxygen species; (2) suppression of GJIC by adherent neutrophils is mediated by ROS in HUVECs, not by proteases, and this suppression is attributed to tyrosine phosphorylation of gap junction protein caused by neutrophil adhesion through integrin and ICAM-1 binding.