In testing the central hypothesis of our previous proposal, that ROS phagocytosis holds in check pro-inflammatory/cell injury signaling in the RPE, we discovered a lipid mediator that accumulates under those conditions and whose bioactivity includes inhibition of cytokine induction of NF-kappaB and COX-2 expression. The novel lipid (that we named neuroprotectin D1, NPD1) also inhibits oxidative stress-triggered RPE apoptosis and provides potent protection against oxidative stress (H2O2/TNFalpha or A2E). NPD1 formation is initiated by phospholipase A2-mediated release of DHA. We also developed a novel PAF antagonist, LAU-0901, that is cytoprotective, and at the same time, up-regulates expression of prosaposin, one of the differentially expressed genes in RPE in the presence of this PAF antagonist. We now propose to test the hypothesis that in the RPE response to pathological insults, there is coordinated/ concerted survival signaling that involves the synthesis of neuroprotectins, expression of prosaposin, and NFkB modulation. ROS phagocytosis upregulates these responses, and while initially activating these events, oxidative stress, if persistent, can overcome survival signaling. We will use cell, molecular, and biochemical approaches that include liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry (LC-PDA-ESI-MS-MS)-based lipidomic analysis. By using primary human RPE cells, ARPE-19 cells, and maculae from rhesus monkeys of various ages, the results may be assessed in the context of both normal function and macular degeneration. We will (1) test the prediction that RPE cell integrity is maintained by growth-factor regulation of the synthesis of NPD1during ROS phagocytosis. These studies will determine how PEDF and other growth factors regulate enzyme-mediated DHA-oxygenation pathways in the RPE and thus contribute to defining mechanisms through which ROS phagocytosis is a trigger of NPD1 synthesis in the RPE; (2) Test the prediction that NPD1 promotes RPE survival during photooxidative damage. A2E and A2E oxiranes (epoxides) are known to accumulate in the aging RPE and to mediate apoptosis in Stargardt disease and AMD. These experiments will specifically test the prediction that A2E-mediated RPE cell apoptosis can be down-regulated by NPD1; (3) Test the hypothesis that NPD1 synthesis is down-regulated in the aging macula. We will study the macula from Rhesus monkeys during aging as well as during ischemia to define its ability to synthesize NPD1 and additional DHA-derived messengers; and (4) Test the prediction that PAF antagonism promotes RPE cell survival. This will include defining the mechanism of prosaposin gene induction in RPE cytoprotection, making use of LAU-0901.
We aim for a better understanding of AMD and to contribute to the development of effective therapies.
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