Visualization of The Uptake of High-density Lipoprotein by Rat Aortic Endothelial Cells and Smooth Muscle Cells in vitro

Abstract

High-density lipoproteins (HDL) were conjugated to Fluorescein 1,1′-dioctadecyl 3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for the investigation of ultrastructural aspects of binding and uptake of HDL by cholesterol-loaded cultured endothelial and smooth muscle cells from rat aorta. When cells were incubated for 2 h at 4°C, HDL–DiI and HDL–gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37°C for 5 min, HDL–DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C₁₂. HDL–gold conjugates appeared in the plasmalemmal invaginations and plasmalemmal vesicles. After incubation for 15 min, most of the HDL–gold conjugates reappeared on the cell surface. After incubation for 30 min, only a few conjugates were observed and they localized in lysosomal-like bodies. Quantitative data indicated that when the cholesterol-loaded cells were incubated at 4°C for 2 h, the numbers of HDL–gold associated in clusters on the endothelial cell surface was 1.18 clusters/μm. When cells were returned to incubation at 37°C for 5 min, this value decreased to 0.7, increased again to 1.13 at 15 min, and decreased to 0.29 at 30 min. The numbers of clusters in the plasmalemmal invaginations were 0.06 clusters/μm at 4°C for 2 h, increased to 0.34 at 37°C for 5 min and decreased gradually to 0.19 and 0.04 at 15 and 30 min, respectively. The incidence of clusters in the plasmalemmal vesicles per non-nuclear cytoplasm was 0.01 clusters/μm² at 4°C for 2 h, increased significantly to 1.08 at 37°C for 5 min, and decreased to 0.43 and 0.14 at 15 and 30 min, respectively. This work supports that the plasmalemmal invaginations and plasmalemmal vesicles are linked to the HDL uptake in cholesterol-loaded aortic endothelial cells and smooth muscle cells.