Abstract
Abstract: :
Purpose:The essential linolenic acid family includes docosahexaenoic acid (DHA). DHA’s highest concentrations are in photoreceptor outer segments, and the RPE is critical for its uptake and recycling. DHA is a major target of lipid peroxidation and is thought to create specific domains for protein function in photoreceptor membranes. By lipidomic analysis we identified novel stereospecific docosanoids derived from enzyme–mediated oxygenation of DHA in ARPE–19 cells and demonstrated their bioactivity. Methods:Oxidative stress–induced apoptosis (H2O2/TNFα) in ARPE–19 cells was assessed by Hoechst staining, DNA fragmentation, and ELISA of mono– and oligonucleosomes. Caspase–3 cleavage was assayed by stable transfection of a construct encoding a peptide containing the caspase–3 cleavage sequence, DEVD, and by exogenous substrate. We used tandem LC–PDA–ESI–MS–MS–based lipidomic analysis to identify the novel stereospecific messengers. Results:ARPE–19 cells activate the biosynthesis of 10,17S–docosatriene, characterized by prominent fragment ions at m/z 359 and other diagnostic ions as reported with the discovery of this new messenger in brain (Marcheselli et al., J. Biol. Chem. 278:43807, 2003). This DHA–oxygenation pathway’s initial RPE metabolite is 17–S–hydroperoxy–DHA. Activators of this biosynthetic route were H2O2/TNFα or IL1ß. Oxidative stress–triggered apoptosis and caspase–3 cleavage were inhibited by 10,17S–docosatriene (50 nM), which also down–regulated IL1ß–induced COX–2 expression. Conclusions:In ARPE–19, DHA is the precursor of novel neuroprotective signaling. 10,17S–docosatriene synthesis is enhanced by cytokines and oxidative stress. This docosanoid potently counteracts pro–inflammatory and cell–damaging responses. These findings are of interest because they elucidate endogenous survival signaling in RPE, and may be useful in the design of new neuroprotective therapeutic strategies for macular degeneration and other retinal diseases. (Support: NIH R01EY05121, P20RR16816–COBRE)
Keywords: cell death/apoptosis • neuroprotection • retinal pigment epithelium