Anti-atherogenic functions of HDL are likely linked to its ability to mediate several steps of the reverse cholesterol transport, including the rate-limiting cellular cholesterol efflux, and to its anti-oxidative and anti-inflammatory effects. Mounting evidence supports the concept that HDL may lose its functionality in some circumstances and disease states, and that HDL dysfunction contributes to the development and progression of atherosclerosis. The goal of our research is to define the mechanisms for HDL dysfunction in three common chronic diseases associated with elevated risk of atherosclerotic cardiovascular disease: Familial Hypercholesterolemia (FH), Rheumatoid Arthritis (RA), and Chronic Kidney Disease (CKD). The driving hypothesis of our proposal is that excessive oxidative burden will impair HDL's ability to regulate tissue levels of oxidative products, reduce its anti-inflammatory properties, and diminish its ability to promote cholesterol efflux from cells such as arterial macrophages. Core B will be an integral part of the PPG by providing both preparative and analytical services for isolation and analysis of lipoproteins, separation and preparation of lipoprotein components, and in vitro assays of HDL function using macrophages and other cell types.
Aim 1 is to provide isolation and analysis of lipoproteins, including HDL subfractions and apoproteins, including apoAl and apoAII, for functional studies and analyses of protein modifications.
Aim 2 is to provide assays for HDL functions, including the promotion of cholesterol efflux from macrophages and the control of inflammation and oxidation using established and validated cell-based and cell-free methods. Our methods to study cellular cholesterol losses takes into account both influx and efflux, and uses actual HDL as cholesterol acceptor. Core B provides a number of services to investigators that require unique instrumentation and methodologies and demand rigorous standardization procedures. It would be costly, inefficient, and cumbersome to establish these procedures in the laboratories of individual PPG investigators. The consolidation of these services into one laboratory provides investigators with efficient, high quality, low-cost analyses. Other objectives of Core B include: 1. To provide education and training to investigators, fellows, and research technicians on the science and methods of lipoprotein preparation and function, and on the best use of and logistical access to the services provided;2. To assist investigators in the development of methodologies a-la-carte, depending on experimental needs not currently foreseen.
This PPG application is based on a reliable and consistent production of reagents such as lipoproteins and monocytes, the uniform conduct of very sensitive functional assays such as cholesterol extraction from loaded cells, and a standardized analysis and interpretation of results. Core B will assure consistency, reliability, and standardization for all procedures and tests. This will fuel cross-pollination and increase cohesiveness and comparability of data among projects.
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