Sergey N. Preobrazhensky

Cholesterol can stimulate secretion of apolipoprotein B by cultured human hepatocytes.

During a 5 day cultivation of human hepatocytes in a primary culture the secretion of apolipoprotein B was measured by enzyme-linked immunosorbent assay. Density-gradient ultracentrifugation demonstrated that the majority of the secreted apolipoprotein B was associated with the very-low-density lipoprotein fraction. Exposure of the cells to cholesterol (5-100 micrograms/ml) resulted in a dose-dependent increase in apolipoprotein B secretion rate.

Effect of cell cholesterol content on apolipoprotein B secretion and LDL receptor activity in the human hepatoma cell line, HepG2

Human hepatoma HepG2 cells were used to study the effects of cholesterol loading and depletion on apolipoprotein B (apoB) secretion and low-density lipoprotein (LDL) receptor activity. Exposure of HepG2 cells to cholesterol and oleic acid, which elevated intracellular cholesterol levels, stimulated apoB secretion and reduced receptor-mediated uptake of LDL, whereas recombinant complexes of apolipoprotein A-I with dimyristoylphosphatidylcholine, which depleted the cellular cholesterol pool, inhibited apoB secretion and up-regulated LDL receptors. Significant negative correlation (r = -0.92, P less than 0.001) between the levels of apoB secretion and LDL uptake was found. These data suggest that the cholesterol content of the cells may induce concomitant changes in apoB secretion and LDL receptor activity.

Study of low density lipoprotein interaction with platelets by flow cytofluorimetry

Low density lipoproteins (LDL), .the main cholesterol carriers in human plasma, are known to interact via the receptor pathway with cultured blood and vessel wall cells. Once bound to the receptor on the cell surface, LDL are internalized and delivered to lysosomes. The LDL degradation in lysosomes results in the release of cholesterol and other lipids, utilized for membrane synthesis [ 1,2]. Platelets were isolated from the plasma of healthy donors by gel filtration on Sepharose 2B [ 1 l] in tyrode solution without Ca2+ and Mg2+, containing bovine serum albumin (3.5 mg/ml, Sigma) and apyrase (0.2 mg/ml, Sigma).

Liposome uptake by cultured macrophages mediated by modified low-density lipoproteins

We have investigated effects of native low-density lipoproteins (LDL) and malondialdehyde-treated LDL on the interaction of 5(6)-carboxyfluorescein-labeled liposomes bearing antibodies to LDL with cultured J774 macrophages. It was found that an addition of modified LDL to the incubation medium resulted in 15-20-fold increase of carboxyfluorescein binding to cells, whereas native LDL did not produce such effect. The increase of carboxyfluorescein binding to macrophages in the presence of modified LDL was not due to an enhanced leakage of the label from liposomes. The modified-LDL-mediated binding of carboxyfluorescein to cells was reduced to 20-30% of the initial level in the presence of cell-respiration inhibitors (NaF and antimycin A). Fluorescent microscopy data also indicate the modified-LDL-induced incorporation of liposome contents into cells. The results obtained in this study make it possible to assume that in the presence of malondialdehyde-treated LDL, liposomes with antibodies to LDL may be incorporated into macrophages via the receptor-mediated pathway for modified LDL.

High-affinity association and degradation of 125I-labelled low density lipoproteins by human hepatocytes in primary culture.

Human hepatocyte LDL Cell association Degradation High affinity association

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous GM1- and GD1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (GM3, GD1a, GT1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (GM1, GM2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 μM concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.

Flow cytometric assay for evaluation of the effects of cell density on cytotoxicity and induction of apoptosis

BACKGROUND We used a flow cytometric assay, which allows us to perform precise measurements within a wide range of cell concentrations to study the effect of the density of cultured cells on their sensitivity to cytotoxic compounds. METHODS To measure cytotoxic action, cells are plated in a 96-well plate at a density ranging from 700 to 100,000 cells/ml and are allowed to grow for 72 h in the presence of various concentrations of a cytotoxic agent. To quantitate the number of surviving cells, each sample is analyzed in a flow cytometer with equal acquisition time. Viable cells are identified by light scattering characteristics identical to those for untreated cells. To estimate the amount of viable, apoptotic, or necrotic (late apoptotic) cells, the samples are stained with Annexin V and propidium iodide. RESULTS Using this method, we found that the cytotoxicity of ascorbic acid for malignant lymphoid CEM-C7 cells can be increased significantly when cell density decreases, reaching a value that is typically lower than the normal physiological concentration of ascorbic acid in blood. CONCLUSION The flow cytometric analysis described in this study can be useful in comparing the effects of cell density on the cytotoxic action of various compounds.

Low-density lipoproteins interact with liposome-binding sites on the cell surface

Under physiological conditions significant amounts of low‐density lipoprotein LDL particles ar taken up by cells independently of specific high affinity LDL receptors (apo‐B receptors). Previously it was established that some cells contain surface sites capable of binding liposomes. We proposed that liposome‐binding sites could contribute to LDL interaction with the cell surface via phospholipid molecules of LDL particles. To check this hypothesis we studied the competitive interaction of human LDL and DPPC liposomes with mouse embryo fibroblasts depleted of apo‐B receptors by preliminary incubation with LDL. We have found that after removal of the liposome‐binding sites from cell lamellae these areas of the cell surface lose their ability to bind LDL.

Interaction of Fluorescently-Labeled Low Density Lipoproteins with Human Aortic Cells in the Primary Culture

The interaction of lipoproteins with vascular wall cells presents particular interest in relation to studies of atherosclerosis pathogenesis. The formation of an atherosclerotic plaque has been now unambigously connected to an excessive accumulation of lipids both in the extracellular space and within the cells. Numerous data obtained from experiments with cell cultures indicate the important role of low density lipoproteins (LDLs) in regulation of the lipid exchange in cells.