Visualization Of Uptake Of High Density Lipoprotein By Rat Aortic Endothelial Cells In Vitro

The high concentration of low-density lipoprotein in the plasma is the major risk factor of atherosclerosis. On the other hand, another plasma lipoprotein—high-density lipoprotein (HDL) — is inversely correlated with atherosclerosis. Recent studies have demonstrated that HDL mediates the transport of cholesterol from peripheral tissues to the liver. However there is considerable debate about the mechanisms by which high density lipoprotein removes excess cholesterol from cells. Two different pathway were suggested: (i) a docking receptor promoting cholesterol translocation, or (ii) a receptor mediated intracellular endosomal pathway termed “retroendocytosis”. In the present study, we performed immunofluorescence and electron microscopy to directly visualize the uptake of HDL using cholesterol-load rat aortic endothelial and smooth muscle cells.Endothelial cells were obtained from rat aorta and cultured in medium under 5% CO2 / 95% air atmosphere. Then confluent monolayers of endothelial cells were incubated in cholesterol for 48 hr. The cells were precooled for 2 hr at 4°C with PBS containing HDL-Dil or HDL-gold (10 nm) at a concentration of 80 j± g protein per ml PBS. Internalization experiments were carried out by incubation at 37°C for 0, 5, 15, and 30 min, followed by three washes with PBS (pH 7.4). Then the cells were processed for fluorescence and transmission electron microscopy.