In this project, we have examined the potential of pathologic conditions of the circulatory system to oxidatively damage the vascular lining known as the endothelium. The endothelium, a single layer of cells interposed between the blood and the tissues, plays a key role in the function of the blood vessel. The endothelium is exposed to oxidants during ischemia/ reperfusion (stroke) and during inflammation. Endothelial cell dysfunction has been linked to diseases such as atherosclerosis and hypertension. Oxidative modifications of proteins occurs when proteins are exposed to oxygen derived-free radicals, causing the introduction of carbonyl groups into the amino acid side chains. Oxidation affects protein function and degradation. To determine the role that protein oxidation plays in endothelial cell damage during oxygen stress is the goal of this project. A tissue culture model of bovine aortic endothelial cells is used to examine the effects of oxidants on protein modification and cellular viability. Cellular damage is assessed by: (1) protein modification, (2) cellular viability, (3) activities of key metabolic enzymes, (4) cellular energy levels. The ability of various antioxidants to prevent cellular damage was also examined. We have found that hydrogen peroxide treatment of endothelial cells causes a time- and dose-dependent loss of viability. Proceeding cytotoxicity, there is a rapid increase in protein oxidation, as well as inactivation of several key metabolic enzymes known to be susceptible to oxidative modification. Energy stores are rapidly lost from the cells, impairing the cell's ability to function. These changes are prevented by a variety of substances, including catalase. It is our hypothesis that oxidation induces cell death by first causing oxidative inactivation of key cellular proteins.
