Visualization of the uptake of high-density lipoprotein by rat aortic endothelial cells and smooth muscle cells in vitro

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 4degreesC, HDL-DiI and HDL-gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37degreesC for 5 min, HDL-DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C-12. 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 4degreesC for 2 h, the numbers of HDL-gold associated in clusters on the endothelial cell surface was 1.18 clusters/mum. When cells were returned to incubation at 37degreesC 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/mum at 4degreesC for 2 h, increased to 0.34 at 37degreesC 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/mum(2) at 4degreesC for 2 h, increased significantly to 1.08 at 37degreesC 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.