All ETDs from UAB

Advisory Committee Chair

Victor M Darley-Usmar

Advisory Committee Members

Shannon Bailey

Scott Ballinger

Janusz Kabarowksi

Aimee Landar

Rakesh Patel

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Free radical catalyzed oxidation of polyunsaturated fatty acids (PUFAs) such as arachidonic acid is increased in cardiovascular disease states, including atherosclerosis and heart failure. Oxidized lipids have been extensively studied and found to recapitulate several key steps in atherogenesis. However, clinical trials with antioxidants such as alpha-tocopherol have been less promising than originally predicted. Now appreciated as more than just biomarkers of disease, lipid peroxidation products have been shown to have roles in pathogenesis as well as physiology. Of particular interest are reactive lipid species that possess electrophilic carbonyls enabling them to act in a receptor-independent manner. To date, the tools for studying protein modification by reactive lipid species have used a candidate approach, focusing on one product at a time and the proteome it modifies. While yielding insight, data from these studies forms an incomplete picture of modifications that occur in vivo, where it is expected that a large number of reactive lipids will be formed simultaneously. For this thesis, we took a different approach, allowing the study of a large number of protein modifications occurring following the endogenous oxidation of tagged derivatives of arachidonic acid. Oxidants relevant to pathology were studied including hydrogen peroxide, myoglobin, and free heme. Heme-mediated lipid peroxidation was found to modify endothelial cell proteins in a thiol-independent manner, yielding insight into the reactivity of the oxidized lipidome in this setting. Mitochondrial localization of BODIPY-arachidonic acid signal was found, signifying that oxidized products of arachidonic acid localize to this organelle following heme exposure. Several proteins were found to be modified within the mitochondrial compartment, one of which was the voltage dependent anion channel (VDAC)--a protein involved in regulating respiration, cell death, and mitophagy. While high concentrations of hemin were found to result in bioenergetic dysfunction that could be protected by alpha-tocopherol, low concentrations of hemin treatment in the same cells resulted in an increase in antioxidant responses, heme oxygenase-1 and glutathione. Design of future therapies could be aimed at inhibiting lipid peroxidation in specific subcellular locales, such as the mitochondria, without inhibiting their modification of critically important cytosolic proteins involved in the antioxidant response.

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