Hypoxia/reoxygenation decreases endothelial glycocalyx via reactive oxygen species and calcium signaling in a cellular model for shock

Abstract

BACKGROUND 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 Ca2+ signaling. METHODS 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 Ca2+ chelator BAPTA or Ca2+ 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. RESULTS 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. CONCLUSION 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 Ca2+ signaling. Notably, we found that ROS are generated upstream of Ca2+, but that ROS-mediated damage to the glycocalyx is dependent on Ca2+.

Effects of graft preservation conditions on coronary endothelium and cardiac functional recovery in a rat model of donation after circulatory death

“…In fact, calcium content correlated positively with cardiac dysfunction and mitochondrial damage, which could be expected, as hyperkalemic cardioplegia can potentially exacerbate Ca 2+ overload and increase reperfusion injury 14 . In addition, calcium content correlated positively with glycocalyx degradation, which is in agreement with Jackson-Weaver et al, 37 who observed that I/R injury promoted Ca 2+ release from endothelial cells, which led to glycocalyx damage in cultured cells.…”

The endothelial glycocalyx in critical illness: A pediatric perspective

“…These oxygen free radicals go on to promote hypoxia-inducible transcription factor expression that may sustain pro-inflammatory cytokine production and signaling [57] . Together, the higher presence of reactive oxygen species and circulating pro-inflammatory milieu [58] during chronic hypoxemia increase the risk of EG thinning [59] , though the impact of chronic hypoxemia on pediatric EG expression has not been systematically evaluated.…”

“…Children who require CPB generally have a congenital heart defect requiring corrective or palliative surgical intervention or need heart and/or lung transplantation. Depending on the type of congenital heart defect or degree of lung disease requiring transplantation, some children live with chronic hypoxia prior to palliative surgery that, as discussed above, can promote pro-inflammatory endothelial cell signaling [201] and endothelial surface layer thinning [59] . Then, with transition to CPB at the time of surgical intervention, regions of the body experience variable states of hypoperfusion and ischemia.…”

A review on the physiological and pathophysiological role of endothelial glycocalyx

“…Glycocalyx disruption results in organ impairment and loss in critically ill patients (Table 2). [ 31 ]…”