research
High density lipoproteins and reverse cholesterol transport
- Publication date
- 9 December 1992
- Publisher
- 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.