High density lipoproteins and reverse cholesterol transport

The e>:periments described in this thesis were performed in order to clarify the mechanism of efflux of cellular cholesterol, a key step in reverse cholesterol transport. It is not known which factors influence the rate of transfer of cholesterol from cells to HDL. Rate limiting steps are important steps in the efflux process and may give more insight into the mechanism of the efflux of cellular cholesterol. Possible rate limiting steps are the cholesteryl ester hydrolysis and subsequent transport of cholesterol to the plasma membrane, the desorption of cholesterol from the membrane, the binding of HDL to the membrane or the composition of the HDL particles. The major pan of the experiments described in this thesis were performed with human endothelial cells (EAhy 926 cell line). These cells are hybrids of human umbilical vein endothelial cells with lung carcinoma cells (A549 line) and show many characteristics of differentiated endothelial cells, e.g., the expression of von Wille brand factor, tissue plasminogen activator, plasminogen activtor inhibitor-type 1, and the production of prostacyclin. In addition, the cells formed confluent monolayers with contact-inhibition of cell growth. For obvious reasons (see section 1.6) we choose to measure only net mass transfer of cholesterol from the cells and first developed a method to enrich the cells with cholesterol using cationized LDL (see chapter 2). Subsequently, we studied the efflux of cholesterol from loaded EAhy 926 cells by different plasma lipoproteins, and the effects of the plasma enzyme lecithin:cholesterol acyltransferase, as described in chapters 3 and 4. In chapter 5 the effect of HDL binding on HDL-mediated cholesterol efflux was studied using modifications of HDL with tetranitromethane and dimethyl suberimidate. The effect of cell membrane phospholipid composition on HDL binding and cholesterol efflux is described in chapter 6. All these experiments were carried out with ultracentrifugally isolated HDL or HDL,. HDL is a heterogeneous population of lipoprotein particles and HDL subfractions with distinct apolipoprotein compositions can be isolated from plasma with immune-affinity columns. The different apolipoprotein composition of these HDL subfractions are expected to be important for the interaction with possible plasma membrane HDL binding proteins and may therefore clarify the role of HDL binding in the efflux of cellular cholesterol. Chapter 7 and 8 contain a description of our investigations of the role of different immunopurified HDL subfractions in the efflux of cellular cholesterol from various cell types as well as in the uptake of HDL hy hepatocytes.

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