The overall goals of this project are: 1) to investigate the role of lipoprotein lipase (LPL) in the pathogenesis of atherosclerosis and determination of elements which regulate its expression in macrophages and 2) to prepare the candidate for a productive career in cardiovascular research by providing him with appropriate training, environment and support. In the first phase of the project, the candidate will examine LPL expression in atherosclerotic lesions by using LPL-specific immunocytochemistry with an antibody and riboprobe which have been developed by the candidate while working in the laboratory of the Primary Sponsor, Dr. Alan Chait. The candidate has demonstrated that LPL mRNA is produced by macrophage-derived foam cells in advanced human coronary atherosclerotic plaques. The antibody and riboprobe for LPL will be used in a well-defined animal model of atherogenesis to examine LPL expression by specific cell types at all stages of lesion development. The expression of apolipoprotein E and of specific cytokines will also be examined to determine their topological correlation with LPL expression. The results of these experiments will be used to design studies planned for the second phase of the project. In the second phase of the project, the candidate will move to the laboratory of the Secondary Sponsor, Dr. Deeb, to work with Dr. Deeb on his already funded project to identify cis-acting elements of the LPL gene responsible for tissue-specific expression. In vitro experiments will be performed to identify potential regulatory sequences of the LPL gene by virtue of their ability to bind to specific transcription factors, as revealed by nuclease hypersensitivity, genomic footprinting and gel mobility retardation assays. In vivo experiments will also be performed in which DNA constructs will be used to investigate regulatory sequences responsible for cell-specific and developmental stage-dependent expression of LPL in transgenic mice. This approach will allow for rapid assessment of expression by using lac Z as a reporter gene, and provide an in vivo system to verify the findings of the planned in vitro experiments. These projects will provide the candidate with experience in advanced molecular biology techniques which will be invaluable for his research career.
Showing the most recent 10 out of 16 publications
