This grant renewal application focuses on understanding the role of lipid-based oxidative protein modification (oxPM) in the etiology of eye diseases. We postulate that oxPM can interfere with protein function or result in pathological receptor-mediated responses. The overarching questions are: (1) how are oxPM generated in vivo, (2) which proteins are modified, (3) what are the pathological consequences, and (4) what therapeutic interventions can prevent oxPM formation or block their receptor-mediated sequelae. Biological entities must adapt to the nonenzymatic reactions, such as free radical-induced lipid oxidation, to which biomolecules are prone. Understanding these proclivities is the cornerstone of our approach to unraveling the involvements of lipid oxidation in disease processes. Our studies have resulted in the identification of many new biologically active oxidized lipids, characterization of their reactions with proteins, and molecular level insights into the pathological consequences of this oxidative biochemistry. One approach, that has been especially productive for us, is to learn about the chemistry of lipid oxidation and the reactions of lipid oxidation products, follow by an investigation of the biological occurrence and consequences of that chemistry. Such studies led to our discoveries of levuglandins (LGs) and isolevuglandins (isoLGs), extremely reactive products of enzymatic and nonenzymatic lipid oxidation, respectively, that are generated through novel rearrangements of endoperoxide intermediates. Because they react within seconds with proteins, these stealthy toxins evaded detection in vivo. But armed with knowledge of their chemistry obtained from """"""""model studies"""""""", we devised methods to detect their protein adducts. Their formation in vivo is now firmly established, and an understanding of their involvement in a host of disease processes is emerging. Our immediate goals for the next four years are to understand: (1) the role of LG and isoLG-based oxPM in the etiology of glaucoma and keratitis, and the utility of vitamin B6 for preventing their formation in vivo, (2) the involvements of carboxyalkyl pyrrole-modified proteins in age-related macular degeneration, choroidal and cornea! neovascularization, and (3) the utility of single-chain antibodies for blocking receptor-mediated pathological responses to oxPM. The understanding gleaned through these studies will provide new approaches to the prevention, early diagnosis, and treatment of eye diseases associated with oxidative injury.
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