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The -3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflammatory properties are poorly understood. DHA is highly susceptible to peroxidation, which yields an array of potentially bioactive lipid species. One class of compounds are cyclopentenone neuroprostanes (A 4 /J 4 -NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show

12,14 -prostaglandin J2 (15d-PGJ2) and 15-J2-isoprostane induce both reactive oxygen species (ROS) formation and cellular antioxidant defenses, such as heme oxygenase-1 (HO-1) and glutathione (GSH). When we compared the ability of these distinct electrophiles to stimulate GSH and HO-1 production, the cyclopentenone electrophiles were somewhat more potent than HNE. Over the concentration range required to observe equivalent induction of GSH, dichlorofluorescein fluorescence was used to determine both the location and amounts of electrophilic lipid-dependent ROS formation in endothelial cells. The origin of the ROS on exposure to these compounds was largely mitochondrial. To investigate the possibility that the increased ROS formation was due to mitochondrial localization of the lipids, we prepared a novel fluorescently labeled form of the electrophilic lipid 15d-PGJ 2. The lipid demonstrated strong colocalization with the mitochondria, an effect which was not observed by using a fluorescently labeled nonelectrophilic lipid. The role of mitochondria was confirmed by using cells deficient in functional mitochondria. On the basis of these data, we propose that ROS formation in endothelial cells is due to the direct interaction of these lipids with the organelle. 15-deoxy-⌬

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Although both inflammation and oxidative stress contribute to the pathogenesis of many disease states, the interaction between the two is poorly understood. Cyclopentenone isoprostanes (IsoPs), highly reactive structural isomers of the bioactive cyclopentenone prostaglandins PGA 2 and PGJ 2 , are formed non-enzymatically as products of oxidative stress in vivo. We have, for the first time, examined the effects of synthetic 15-A 2 -and 15-J 2 -IsoPs, two groups of endogenous cyclopentenone IsoPs, on the inflammatory response in RAW264.7 and primary murine macrophages. Cyclopentenone IsoPs potently inhibited lipopolysaccharide-stimulated IB␣ degradation and subsequent NF-B nuclear translocation and transcriptional activity. Expression of inducible nitric-oxide synthase and cyclooxygenase-2 were also inhibited by cyclopentenone IsoPs as was nitrite and prostaglandin production (IC 50 ϳ 360 and 210 nM, respectively). 15-J 2 -IsoPs potently activated peroxisome proliferator-activated receptor ␥ (PPAR␥) nuclear receptors, whereas 15-A 2 -IsoP did not, although the anti-inflammatory effects of both molecules were PPAR␥-independent. Interestingly 15-A 2 -IsoPs induced oxidative stress in RAW cells that was blocked by the antioxidant 4-hydroxy-TEMPO (TEMPOL) or the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. TEMPOL also abrogated the inhibitory effect of 15-A 2 -IsoPs on lipopolysaccharide-induced NF-B activation, inducible nitricoxide synthase expression, and nitrite production, suggesting that 15-A 2 -IsoPs inhibit the NF-B pathway at least partially via a redoxdependent mechanism. 15-J 2 -IsoP, but not 15-A 2 -IsoP, also potently induced RAW cell apoptosis again via a PPAR␥-independent mechanism. These findings suggest that cyclopentenone IsoPs may serve as negative feedback regulators of inflammation and have important implications for defining the role of oxidative stress in the inflammatory response.