The applicant has trained clinically in internal medicine and cardiology. The proposed work represents further training necessary for the development of her career as an independent investigator in molecular cardiology. Techniques such as the synthesis and use of photoaffinity labels, enzyme and lipid assays, and immunoblot and monoclonal antibody technology will be used to develop an understanding of basic molecular events underlying intracellular cholesterol metabolism in vascular tissue, such as those occurring after interaction of cells with messengers such as LDL. A key gap in our understanding of such events has been lack of identification of acyl CoA-cholesterol-O-acyltransferase (ACAT) and thus lack of knowledge concerning the regulation of this important enzyme in the cholesterol ester cycle. The immediate goal of this project is the identification of ACAT, which will be accomplished using derivatives of a synthesized 125I labeled photoreactive fatty acid analog to photoaffinity label the enzyme. Subsequently, quantitation and characterization of ACAT will be performed and the developed techniques will be applied to the study of ACAT and its regulation in vascular tissue and cell culture systems. The major focus of this work is to understand the molecular events regulating intracellular cholesterol metabolism in vascular tissue, especially those occurring after interaction of cells with messengers such as LDL. The applicant will work under the supervision of Dr. Louis Lange, an acknowledged leader in the enzymology and biochemistry of neutral lipids, who provides a strong role model both in the basic science disciplines and in the clinical arena. His laboratory is fully equipped for the described experimental work and also provides a challenging and supportive intellectual environment with ties to both the Pharmacology and Pathology Departments at Washington University School of Medicine. The applicant will be working with the Division of Cardiology with 85 to 90% of her time devoted to basic research and 10 to 15% to clinical work throughout the period of the award.
