Endothelial dysfunction after hypoxia-reoxygenation: do in vitro models work?

Hypoxia-reoxygenation (H/R) causes tissue injury, mainly due to free radical production and leukocyte activation. H/R-induced endothelial damage is widely described, however in pharmacological research, there are only sporadic functional studies investigating in vitro vascular H/R. This methodological study compares results of in vivo and in vitro functional experiments. In canine and porcine in vivo experiments hearts were subjected to regional or global ischemia and reperfusion. Blood flow was measured on the left anterior descending coronary artery with a perivascular ultrasonic probe. Endothelium-dependent and -independent vasodilation was assessed after single-bolus intracoronary administration of acetylcholine and sodium nitroprusside (SNP). In organ bath experiments, isolated porcine coronary and rat aortic rings were investigated. Hypoxia (30, 45, 60, 120 min) was induced in the chamber by gassing with 95% N2-5% CO2. (pO2 < 30 mm Hg) During the subsequent reoxygenation (30 min), gassing was changed to 95% O2-5% CO2. The dose-dependent vasoresponse to acetylcholine, bradykinin and SNP was investigated in precontracted rings under normoxic conditions and after H/R. Endothelial function assessed by coronary blood flow measurements was impaired after ischemia-reperfusion in vivo. Although the typical hypoxic vasomotor response could be observed in vitro, no impairment of endothelial function could be proven after H/R in any groups. We conclude that endothelial injury occurring in vessel rings during in vitro H/R is too slight (probably due to lack of activated leukocytes) and cannot be demonstrated in functional measurements. Therefore the experimental model of in vitro vascular H/R is not suited for reliable investigation of pharmacological attempts.

Endothelial dysfunction after long-term cold storage in HTK organ preservation solutions: effects of iron chelators and N-alpha-acetyl-L-histidine.

BACKGROUND: To improve the rate of successful heart transplantations, organ preservation should be optimized in cardiac transplantation surgery. Iron-dependent oxidative damage and iron-independent, chloride-dependent injury of the endothelium have been described after cold ischemic storage and reperfusion, leading to an enhanced rate of complications and unfavorable outcomes. This screening study tested the effects of iron chelator supplementation in different histidine-tryptophan-ketoglutarate (HTK) organ preservation solutions on endothelial function in a long-term storage model of the isolated rat aorta. METHODS: Isolated rat aortic rings underwent a 24-hour cold ischemic preservation in different HTK solutions supplemented with iron chelators of low (deferoxamine) and high (LK-614) membrane permeability. In vascular reactivity measurements we investigated the phenyleprine-induced contraction and both endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine and sodium nitroprusside with and without an additional external oxidant injury during re-oxygenation. RESULTS: Traditional HTK solution, Custodiol, failed to prevent endothelial dysfunction in our experiments. Use of chloride-poor HTK solutions containing N-alpha-acetyl-l-histidine with and without supplementation with LK-614, but not with deferoxamine, resulted in significant improvement of impaired endothelial function; moreover, complete protection of the endothelium was feasible after 24-hour cold storage. Endothelium-independent functions of vascular smooth muscle were not affected in any of the groups. CONCLUSIONS: Our results demonstrate the important pathophysiologic role of iron-dependent oxidative injury in the development of endothelial dysfunction after cold storage, which can be prevented by cell-permeable iron chelators.