Lipid peroxidation and modification of lipid composition in an endothelial cell model of ischemia and reperfusion

Among the changes that accompany the development of ischemia are alterations in the composition and turnover of membrane phospholipids. To study these effects: a cell culture model was developed to facilitate accurate measurements of lipids over varying Intervals of ischemia and reperfusion (I/R). In order to mimic ischemia, rabbit aortic endothelial cells were grown to concurrency on collagen coated beads and the bead cultures allowed to settle to the bottom of a conical test tube or spectrofluorometric cuvette. The cell-coated beads were then resuspended in media to simulate the process of reperfusion, Survival after ischemia/reperfusion, was determined by measurements of cellular replating efficiency, and found to decrease sifter periods longer than three hours of ischemia (followed by 24 h of reperfusion). Plating efficiencies were reduced to nearly 50% after 5 h of ischemia followed by reperfusion Release of LDH inversely correlated with cell survival, and lactate production, ATP levels, and extracellular H2O2 concentration were all affected by the duration of ischemia. These changes could be directly related to rates of cellular oxygen consumption which decreased by 50% after 5 h of ischemia, while the percentage of oxygen consumption not be inhibitable by cyanide, increased, Release of esterified fatty acids, which was partly inhibited by the phospholipase A(2) inhibitor, mepacrine, was stimulated by increasing periods of ischemia while the incorporation of free fatty acids into phospholipids was inhibited, The incorporation of arachidonic acid was inhibited to a lesser degree than that of oleic or Linoleic acids with a resulting change in phospholipid fatty acyl composition favoring greater proportions of unsaturated fatty acids. In some experiments, the effects of vitamin E or ascorbic acid administered prior to ischemia were studied, The degree of fatty acid unsaturation, fatty acid incorporation into phospholipids, and release from phospholipids into the free fatty acid pool during: ischemia/reperfusion were not affected by prior administration of vitamin E or ascorbic acid. However, the extent of lipid peroxidation during ischemia was inhibited by 100 mM ascorbic acid when present during the ischemia/reperfusion period, but not by vitamin E administered for 24 h prior to ischemia, Ascorbic acid treatment, but not vitamin E, also enabled cells to recover substantial amounts of the ATP lost following prolonged ischemia. The ATP recovery corresponded to an increased cell survival and decreased Lipid peroxidation. Progressive intervals of ischemia followed by reperfusion result in compromised cell respiratory activity and decreased ATP production, and decreased phospholipid acylation leading to net hydrolysis. The associated changes in phospholipid composition, and specifically increased unsaturation appear to favor peroxidation of membrane phospholipids. (C) 1997 Elsevier Science Inc.