Trafficking of HDL and its lipids in mouse models

Cholesterol transport via high density lipoprotein (HDL) is an essential regulatory mechanism to remove excess cholesterol from peripheral tissues and to deliver it back to the liver for disposal. In contrast to the well-described low density lipoprotein (LDL) receptor pathway, in which the entire LDL particle is degraded by the cell, HDL delivers only its core lipids to the cell, termed selective cholesteryl ester uptake. Beside this also HDL particle uptake and transcytosis were described. Diaminobenzidine (DAB)-photooxidation is utilized to convert fluorescent signals into an electron dense precipitate visible electron microscopically. We have recently established this method to visualize HDL and HDL-derived lipids in tissue culture cell lines at an ultrastructural level using fluorophores tracing the protein part of the HDL particle and both fluorescent cholesterol and cholesteryl ester, namely a novel Bodipy-cholesterol and Bodipy- cholesteryl ester. Here we propose to track the protein and lipid part of HDL in mice. We will use immunofluorescence microscopy and electron microscopic imaging techniques to demonstrate the membrane areas involved in HDL binding and lipid unloading in liver. We hypothesize that HDL and its lipid components are handled differently in different tissues. Besides lipid exchange at the cell surface, HDL particle uptake might operate in specialized cells to supply the tissues with the cholesterol needed. Additionally, HDL transcytosis might occur in endothelial cells for instance to supply the atherosclerotic plaque with cholesterol accepting HDL. The project aims to visualize the trafficking of the HDL particle and its lipid constituents which are transferred to tissues during cell association of HDL and the fate of the lipids after unloading. We will compare the HDL uptake pathway to that of LDL and its constituents/lipids to work out the characteristics of HDL metabolism in a more general setting. Besides liver we will assess HDL binding and lipid transport to steroidogenic tissues, kidney and endothelial cells and plaques. Finally, we will analyze HDL binding and lipid unloading in genetically modified mice and mouse models for atherosclerosis to delineate alternative pathways that might be upregulated when the main uptake routes like LDL receptor-mediated endocytosis are impaired or overloaded. We expect that these results will give a detailed picture of the interplay of the major lipoprotein pathways like receptor-mediated endocytosis and selective cholesteryl ester uptake and alternative pathways like retroendocytosis and transcytosis of HDL.

Cholesterol is an essential membrane constituent of all mammalian cells, however excess cholesterol can be toxic. Therefore cells need to keep a tight balance between synthesis, uptake and export, as cholesterol itself cannot be degraded in humans. Cellular cholesterol is transported to acceptors in the blood stream, so-called high density lipoproteins (HDLs), which accept excess cholesterol from peripheral cells and tissues and transport it back to the liver for disposal into the bile. This pathway is called reverse cholesterol transport. Imbalance in cholesterol homeostasis is caused by increased food intake or by genetic factors resulting in disposal of cholesterol at the arterial wall leading to atherosclerosis and subsequently to heart attack, the number one killer in the westernized world. Thus to prevent heart attacks one needs to understand the underlying pathological processes of atherosclerosis.In this project we aimed to track the fate of the HDL particle and its derived lipids. We have recently established a method to visualize HDL and HDL-derived lipids using fluorophores tracing both spherical HDL particles as a whole and its lipid constituents using fluorescent cholesterol and cholesteryl ester. First we showed that the HDL particle needs to integrate with the plasma membrane for cargo transfer. In the liver HDL uptake was decreased by bile acids, however cholesterol transfer increased. In contrast the transfer of cholesterol from hepatic cells to HDL was impaired by cellular stress of the endoplasmic reticulum. The processes in the vessel wall, demonstrated in our experiment in endothelial cells seems to be different, where HDL transcytosis was seen.