Glutathionylated Products of Radical-Induced Lipid Oxidation in Inflammatory Disease

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Limited knowledge of the structures and biological activities of products generated by free radical-induced lipid oxidation in the retina is a barrier to progress in the development of clinically useful new disease markers and mechanistically informed therapeutic interventions. Understand- ing cellular responses to free radical-induced lipid oxidation products is complex owing to their diversity and ability to mimic enzymatically generated receptor agonists. Their unrecognized generation and biological activities almost certainly contribute to the mediocre efficacy of currently available therapeutic measures for age related macular degeneration (AMD). We discovered glutathione (GSH) derivatives produced from oxidatively truncated arachidonyl phospholipids that are structural and functional analogues of cysteinyl (Cys) leukotrienes (LTs). We refer to them as pseudo (ø)LTs. We will test the hypotheses that øLTs contribute to retinal pathology and physiology in a rat model of light-induced retinal degeneration. Pilot studies show that øLTs are present in human retina, are produced in vivo consequent to oxidative insult, and exhibit LT-like biological activities. Therefore, øLTs can elicit cellular responses erroneously presumed to be induced exclusively by CysLTs. Consequently, they are a confounding factor for interpreting previous studies on the involvements of LTs in retinal pathology and physiology. The proposed research will test the hypotheses that øLTs and related GSH derivatives of oxidatively truncated docosahexaenoyl phospholipids contribute to receptor-dependent inflammatory cytokine signal- ing, retinal edema, pathological neovascularization, or physiologically important initiation of retinal pigment epithelium (RPE) autophagy. The potential pathophysiological significance of these glutathionylated products of lipid oxidation lies in the possibility of their ubiquitous generation in high levels, e.g., compared to those of CysLTs. We will test the conclusion of preliminary studies that øLTs are LT receptor agonists and determine if their ability to induce the expression of IL-13 and TGF-b1 promotes LT biosynthesis. Because cross reactivity may confound the interpretation of immunoassays, we quantitate øLTs and LTs by LC-MS/MS. We will determine signaling path- ways, e.g., with RNA-Seq studies, activated by glutathionylated products of free radical-induced lipid oxidation addressing the questions: do they promote inflammation in ocular pathology or initiate autophagy in RPE cells and how do they do it? Molecular level insights into the role of oxidative stress in the pathogenesis of inflammatory eye diseases such as AMD can facilitate development of therapeutic interventions that ameliorate the progression of these common but disabling diseases.