t-butyl hydroperoxide and oxidized low density lipoprotein enhance phospholipid hydrolysis in lipopolysaccharide-stimulated retinal pericytes

Free radicals induced by organic peroxides or oxidized low density lipoprotein (oxLDL) play a critical role in the development of atherosclerosis. In investigating this process, and the concomitant inflammatory response, the role of pericytes, cells supporting the endothelial ones in blood vessels, has received little attention. In this study we tested the hypothesis that tert-butyl hydroperoxide (t-BuOOH) and oxLDL, administered in sublethal doses to the culture medium of retinal pericytes, function as prooxidant signals to increase the stimulation of the peroxidation process induced by lipopolysaccharide (LPS). Confluent cell monolayers were exposed to t-BuOOH (25-400 muM), native LDL or oxLDL (3.4-340 nmol hydroperoxides/mg protein, 1-100 muM). LPS (1 mug/ml), t-BuOOH (200 muM), and oxLDL (100 muM), but not native LDL, incubated for 24 h with cells, markedly increased lipid peroxidation, cytosolic phospholipase A(2) (cPLA(2) activity and arachidonic acid (AA) release in a time- and dose-dependent manner. AACOCF(3), a potent cPLA(2) inhibitor, and the antioxidant alpha -tocopherol strongly inhibited the prooxidant-stimulated AA release. Long-term exposure to maximal concentrations of t-BuOOH (400 muM) or oxLDL (100 muM) had a sharp cytotoxic effect on the cells, described by morphological and biochemical indices. The presence of t-BuOOH or oxLDL at the same time, synergistically increased phospholipid hydrolysis induced by LPS alone. 400 muM t-BuOOH or 100 muM oxLDL had no significant effect on the stimulation of an apoptosis process estimated by DNA laddering and light and electron microscopy. The results indicate that (i) pericytes may be the target of extensive oxidative damage; (ii) activation of cPLA(2) mediates AA liberation; (iii) as longterm regulatory signals, organic peroxide and specific constituents of oxLDL increase the pericyte ability to degrade membrane phospholipids mediated by LPS which was used, in the present study, to simulate in vitro an inflammatory burst in the retinal capillaries. (C) 2001 Published by Elsevier Science B.V.