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Jackson-Weaver, Olan; Friedman, Jessica K; Rodriguez, Laura A; Hoof, Marcus A; Drury, Robert H; Packer, Jacob T; Smith, Alison; Guidry, Chrissy; Duchesne, Juan C
The journal of trauma and acute care surgery, 11/2019, Volume: 87, Issue: 5Journal Article
Ischemia/reperfusion injury (IRI) has been shown to cause endothelial glycocalyx (EG) damage.Whether the hypoxic/ischemic insult or the oxidative and inflammatory stress of reperfusion plays a greater part in glycocalyx damage is not known. Furthermore, the mechanisms by which IRI causes EG damage have not been fully elucidated. The aims of this study were to determine if hypoxia alone or hypoxia/reoxygenation (H/R) caused greater damage to the glycocalyx, and if this damage was mediated by reactive oxygen species (ROS) and Ca signaling. Human umbilical vein endothelial cells were cultured to confluence and exposed to either normoxia (30 minutes), hypoxia (2% O2 for 30 minutes), or H/R (30 minutes hypoxia followed by 30 minutes normoxia). Some cells were pretreated with ROS scavengers TEMPOL, MitoTEMPOL, Febuxostat, or Apocynin, or with the Ca chelator BAPTA or Ca channel blockers 2-aminoethoxydiphenyl borate, A967079, Pyr3, or ML204. Intracellular ROS was quantified for all groups. Endothelial glycocalyx was measured using fluorescently tagged wheat germ agglutinin and imaged with fluorescence microscopy. Glycocalyx thickness was decreased in both hypoxia and H/R groups, with the decrease being greater in the H/R group. TEMPOL, MitoTEMPOL, BAPTA, and 2-aminoethoxydiphenyl borate prevented loss of glycocalyx in H/R. The ROS levels were likewise elevated compared with normoxia in both groups, but were increased in the H/R group compared with hypoxia alone. BAPTA did not prevent ROS production in either group. In our cellular model for shock, we demonstrate that although hypoxia alone is sufficient to produce glycocalyx loss, H/R causes a greater decrease in glycocalyx thickness. Under both conditions damage is dependent on ROS and Ca signaling. Notably, we found that ROS are generated upstream of Ca, but that ROS-mediated damage to the glycocalyx is dependent on Ca.
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