Infrarenal Aortic Occlusion Causes Endothelial Injury via Mitochondrial Reactive Oxygen Species

The endothelial glycocalyx is an anti-inflammatory, anti-coagulant coating of endothelial cells which participates in many physiologic processes. Damage to the glycocalyx occurs secondary to several disease states, and is especially pronounced in ischemia-reperfusion (I/R) injury. In models of trauma and hemorrhage, this damage has been shown to occur secondary to disordered succinate metabolism leading to increased mitochondrial reactive oxygen species (mitoROS). Aortic occlusion has also been associated with I/R injury to the vascular endothelium, though the precise mechanisms by which this occurs, and whether that injury includes glycocalyx shedding, are largely unknown. Consequently, very few therapeutic strategies for protection of the glycocalyx have been investigated. The aim of this study was to determine whether an experimental model of infrarenal aortic occlusion, with or without hemorrhage, causes a mitochondrial ROS-dependent glycocalyx shedding, and at what point this shedding predominantly occurs. We additionally aimed to determine whether plasma succinate was elevated following aortic occlusion.

Male Sprague Dawley rats were anesthetized and laparotomy performed. A jugular catheter was placed and a baseline blood sample collected. In the hemorrhage group, 3 mL of blood was drawn from the jugular catheter. The infrarenal abdominal aorta was isolated and clamped with an atraumatic vascular clamp. Thirty minutes after clamping a second blood sample was collected, followed by unclamping. Blood samples were collected 2 and 15 min after reperfusion. Blood was centrifuged, and succinate and glycocalyx-component syndecan-1 were measured in the plasma using an ELISA. All rats in the treatment arm received identical hemorrhage, clamping, and blood sampling, but were treated with mitochondrial-targeted ROS scavenger mitoTEMPOL prior to clamping the aorta. After euthanizing the rats, calf muscle of the hemorrhage group was flash frozen, sectioned, and stained for glycocalyx with wheat germ agglutinin.

Plasma syndecan-1 levels were significantly (P < 0.05) elevated 2 mins after unclamping the aorta compared to baseline in both groups. Levels remained increased, though not statistically significantly, at 15 min after reperfusion. No significant increase in syndecan-1 was seen after 30 min of ischemia. MitoTEMPOL treatment prevented an increase in plasma syndecan-1. Correspondingly, glycocalyx staining intensity in mitoTEMPOL-treated rats was increased compared to control. Plasma succinate was significantly elevated at 15 min after reperfusion and was not affected by treatment with mitoTEMPOL.

To our knowledge this is the first study which demonstrates a successful therapeutic strategy in the treatment of endothelial glycocalyx shedding caused by aortic occlusion and hemorrhage in an animal model. Our study shows that glycocalyx shedding is attributable to mitochondrial ROS, and occurs following distal reperfusion rather than occlusion of the aorta or during hemorrhage. Consistent with findings in hemorrhagic shock, plasma succinate levels are elevated as a result of aortic occlusion and are upstream of ROS. Taken together, our findings suggest that in the setting of aortic occlusion, a therapeutic intervention could be performed prior to reperfusion which would substantially reduce injury to the vascular endothelium.