Date of Degree
PhD (Doctor of Philosophy)
Khalid N. Kader
Development of an in vitro model for the early stages of cardiovascular disease is a current necessity. Cardiovascular disease is the leading cause of death in the United States and throughout the world. Oxidative stress and reactive oxygen species have been implicated in cardiovascular disease development. An in vitro model of these processes will improve our understanding of cardiovascular disease development and allow for the development of additional treatments.
Atherosclerosis is an inflammatory disease and increased levels of H2O2 are associated with inflammation. The model focuses on H2O2-induced oxidative stress under static and shear conditions. Previous studies have documented increased O2.- and increased cytotoxicity in smooth muscle cells exposed to H2O2.
Under static culture, endothelial cells exposed to H2O2, exhibited increased O2.- over basal levels via NOS and NAPDH oxidase pathways. Increased O2.- was attenuated by MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. Endothelial cell cytotoxicity was increased with H2O2 exposure. The increase in cytotoxicity was diminished upon exposure to Tiron or L-NAME.
Under shear conditions (8.2 dynes/cm2), endothelial cells exposed to H2O2 exhibited increased O2.- compared to control via an L-NAME (specific inhibitor NOS) and Apocynin (NADPH oxidase inhibitor) inhibitable mechanism. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. The increased O2.- was attenuated with MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron (an O2.-scavenger). Endothelial cell attachment under shear with exposure to H2O2 was improved with MnSOD adenoviral-mediated upregulation as observed by decreased loss of the endothelial cell monolayer compared with H2O2 exposed endothelial cells.
Endothelial cells exposed to H2O2 exhibit increased O2.-, suggesting that H2O2-induced oxidative stress may be a reasonable model for atherosclerosis. NOS and NADPH oxidase co-inhibition under shear and static culture demonstrated that NOS and NADPH oxidase inhibition is non-additive under static culture, yet additive under shear. Co-inhibition results suggest a complex relationship between the two enzymes that requires additional experimentation to deconvolve.
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Copyright 2004 Christian Hannon Coyle
Coyle, Christian Hannon. "Mechanisms of H2O2-induced oxidative stress in endothelial cells." dissertation, University of Iowa, 2004.