Unlabeled Low Density Lipoprotein Research Articles

Effect of 4-aminopyrazolo[3,4- d]pyrimidine on the catabolism of high-density lipoprotein apolipoprotein A-I in the rat

The in vivo turnover and sites of catabolism of O-(4- diazo-3-[ 125I] iodobenzoyl) sucrose- labelled rat highdensity lipoprotein (HDL) apolipoprotein A-I were studied in rats treated for 3 days with 4-aminopyrazolo-[3,4- d]pyrimidine (4 APP). It was found that 4APP treatment decreases the serum cholesterol concentration to 6 mg/dl and stimulates the serum decay of labelled HDL. The latter effect could be attributed to an increased catabolism of radioactive HDL apolipoprotein A-I by the liver. When the serum cholesterol concentration was raised to physiological levels by a bolus injection of unlabelled rat HDL, at the time of administration of the labelled HDL, the serum decays and the tissue uptakes of apolipoprotein A-I labelled HDL were identical in 4APP-treated rats and control animals. When a bolus injection of unlabelled human low-density lipoprotein (LDL) was administered to 4APP-treated rats, the serum decay and tissue uptake of apolipoprotein A-I labelled HDL remained rapid. The recovery of radioactivity in the adrenal glands was increased almost 10 fold by 4APP treatment, a phenomenon which was reversed by a bolus injection of unlabelled HDL, but not by injection of unlabelled LDL. It is concluded that treatment of rats with 4APP does not affect the rate of catabolism of rat HDL apolipoprotein A-I, when the serum HDL concentration in the treated animals is restored to physiological levels.

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of β-hydroxy-β-methylglutaryl-CoA reductase and acyl-CoA: cholesterol acyltransf erase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.

Properties of low density lipoprotein binding by cultured swine granulosa cells.

The properties of low density lipoprotein (LDL) binding to ovarian cells were investigated in cultured swine granulosa cells in serum-free monolayer cultures. Swine and human LDL bound with high affinity and specificity, with apparent dissociation constant (Kd) values for swine and human LDL of 1.1 and 3.6 micrograms/ml at 4 C, and 2.7 and 4.8 micrograms/ml at 37 C. In contrast, high density lipoprotein competed sparingly with [125I]iodo-LDL with an apparent half-maximally inhibitory concentration of 650 micrograms/ml. Binding of LDL was dependent upon arginine residues within the apoprotein B moiety, since covalent modification of LDL with 1,2-cyclohexanedione markedly reduced its ability to compete for binding or degradation and to support progesterone biosynthesis. This specific, high affinity saturable binding of LDL to pig granulosa cells exhibited a maximal binding capacity of 0.7 micrograms LDL protein/mg DNA, which corresponds to approximately 5500 LDL receptors per cell. The relative time course of LDL binding, internalization, and degradation by swine granulosa cells was assessed by examining trypsin-releasable (surface-bound) and trypsin-resistant (internalized) [125I]iodo-LDL. At 37 C, granulosa cells exhibited a rapid increase in surface-bound lipoprotein, followed by delayed but subsequently progressive increases in internalized and degraded LDL. LDL degradation by swine granulosa cells was a saturable, temperature- and time-dependent process, with half-maximal degradation occurring at a concentration of 16 micrograms/ml LDL. This correlates closely with the half-maximal concentration of LDL that stimulates progesterone secretion. Degradation of [125I]iodo-LDL was not attributable to bulk fluid-phase pinocytosis, since the cellular ingestion of an impermeant probe, [125I]iodo-polyvinylpyrrolidone, occurred at 1/40 the rate of lipoprotein degradation. In addition, degradation of [125I]iodo-LDL was specifically inhibited by excess unlabeled LDL, decreased by prior exposure of granulosa cells to the soluble sterol, 25-hydroxycholesterol, and antagonized by the lysosomotrophic agent, chloroquine. Moreover, in separate experiments, rates of degradation of LDL were found to be significantly correlated with progesterone one production (r = +0.88, P less than 0.01). In summary, swine granulosa cells exhibit specific, high affinity, saturable, and low capacity receptors for homologous and heterologous LDL. These LDL recognition sites on ovarian cells depend upon cyclohexanedione-sensitive arginine residues with the apoprotein B moiety for effective binding, internalization, and functional (steroidogenic) responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of high density lipoprotein binding to human adipocyte plasma membranes.

Freshly isolated human adipocytes showed specific uptake of 125I-labeled human high density lipoprotein (HDL2 and HDL3), a portion of which could be released by subsequent incubation with excess unlabeled ligand. To study the mechanism of HDL binding, sucrose gradient-purified adipocyte plasma membranes were incubated with radioiodinated lipoprotein particles under equilibrium conditions in the absence (total binding) or presence (nonspecific binding) of 100-fold excess unlabeled ligand. Specific binding of HDL2 and HDL3, calculated by subtracting nonspecific from total binding, was Ca++ independent, unaffected by EDTA, and not abolished by pronase treatment of the membranes. Modification of HDL3 by reductive methylation or cyclohexanedione treatment also failed to affect its binding to adipocyte plasma membranes. High salt concentration (200 mM NaCl) inhibited specific binding of HDL2 and HDL3 but had no effect on LDL binding. A significant portion of 125I-HDL2 or 125I-HDL3 binding was consistently inhibited by adding excess unlabeled LDL, but this inhibition was incomplete as compared with a similar molar excess of unlabeled HDL2 or HDL3. The role of apoproteins (apo) in HDL binding to adipocyte membranes was examined by comparing binding of HDL2 and HDL3 isolated from normal, abetalipoproteinemic (abeta) and apo E-deficient (apo E0) plasma. Specific binding was observed with all normal and mutant HDL particles. Furthermore, a significant portion (61-78%) of abeta-HDL2, apo E0-HDL2, and apo E0-HDL3 binding was inhibited by adding 100-fold excess of unlabeled low density lipoproteins (LDL). The cross-competition of LDL and HDL binding was confirmed by the ability of normal, abeta, and apo E0-HDL2 to completely inhibit 125I-LDL binding. These data suggest that HDL binding is independent of apo E and that the responsible apoprotein(s) of HDL complete with LDL-apo B for binding to the same or closely related site in the adipocyte plasma membrane. Normal and apo E0-HDL3 binding was also completely inhibited by normal HDL2, which suggested that HDL2 and HDL3 probably bind to the same site. Scatchard analysis of normal HDL2, normal HDL3, and apo E0-HDL3 binding data best fitted a one-component binding profile with similar equilibrium dissociation constants (40-96 nM). HDL3 binding was found to be effectively inhibited by anti-human apo AI or anti-human apo AII, but not by anti-human apo B antisera. This binding was also unaffected by monoclonal anti-human apo B or E antibodies known to inhibit binding of apo B or apo E containing lipoprotein to the LDL receptor of cultured fibroblasts. These findings, taken together, suggest that human fat cells possess HDL binding sites with apo AI and /or apo AII specificity. The significant but partial inhibition of HDL2 and HDL3 binding by LDL along with the complete inhibition of LDL binding by HDL2 and HDL3 tends to exclude a single binding site that interacts both lipoproteins and favors the interpretation that LDL and HDL particles bind to multiple recognition sites or to different conformation of the same lipoprotein binding domain on the human fat cell.

Visualization of the hepatic low-density lipoprotein receptor in rats by sequential scintiscans

The usefulness of a gamma camera system for external imaging of the degradation sites of low-density lipoprotein (LDL) is demonstrated in rats. [ 131I]LDL was injected intravenously in normal rats and rats pretreated with 17α-ethinyl estradiol for LDL receptor induction. Distribution of the radioligand was followed for 20 min, for perfusion control [ 131I]albumin was administered. Rats pretreated with 17α-ethinyl estradiol showed a marked increase in the LDL uptake by the liver, which was not competed by the preinjection of physiological amounts of unlabelled LDL. It is concluded that the injection of small amounts of [ 131I]LDL is sufficient to image the hepatic LDL receptor and that the endogenous LDL does not compete effectively at physiological LDL levels.

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

Catabolism of homologous low density lipoproteins (LDL) was studied in primary culture of human hepatocytes (HH). The cell association and degradation of 125I-labeled LDL ( 125I-LDL) were curvilinear functions of substrate concentration. Cell association and degradation of 125I-LDL were inhibited by excess unlabeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-LDL for cell association and degradation. Preincubation of HH with unlabeled LDL caused a 63% inhibition of the 125I-LDL degradation. It is concluded that the catabolism of LDL by HH proceeds in part through a receptor-mediated pathway similar to that demonstrated on extrahepatic cells. Human hepatocyte LDL Cell association Degradation High affinity association

In vivo catabolism of human low density lipoprotein in the rat is mediated by a nonsaturable, low-affinity mechanism

The degradation of human low density lipoprotein (LDL) was analyzed in fasted rats treated for 3 days with either 4-aminopyrazolo-(3,4-d)pyrimidine (4APP) or saline. Treatment with 4APP caused an 80% decrease in serum cholesterol concentration. The mono-exponential serum decay of a tracer amount of labelled LDL was changed neither by 4APP treatment, nor by the simultaneous injection of a bolus of unlabelled LDL. The sites of degradation of human LDL were determined using the nondegradable labelling compound O-(4-diazo-3-[ 125I]iodobenzoyl)sucrose (D 125IBS). The sites of degradation and the rate of degradation of D 125IBS labelled LDL were also not affected by 4APP treatment or by injection of a bolus of unlabelled LDL. It is concluded that human LDL is catabolised in the rat by way of a nonsaturable, low-affinity mechanism. Low density lipoprotein 4-Aminopyrazolo-(3,4-d)pyrimidine Catabolic site LDL receptor B/E receptor O-(4-Diazo-3-[ 125I]iodobenzoyl)sucrose

Metabolism of low-density lipoprotein from patients with diabetic hypertriglyceridemia by cultured human skin fibroblasts.

To test whether triglyceride-enriched low-density lipoprotein (LDL) obtained from subjects with diabetic hypertriglyceridemia is metabolized normally by cells, LDL was separated from seven healthy control subjects (fasting plasma glucose [FPG] 91 +/- 10 mg/dl [mean +/- SD], triglyceride [TG] 110 +/- 47 mg/dl), six diabetic normolipidemic patients (FPG 218 +/- 65 mg/dl; TG 139 +/- 75 mg/dl), six diabetic hypertriglyceridemic patients (FPG 214 +/- 71 mg/dl; TG 1915 +/- 1680 mg/dl), and five nondiabetic hypertriglyceridemic patients (FPG 92 +/- 8 mg/dl; TG 2013 +/- 1889 mg/dl). Binding of 125I-labeled LDL from hypertriglyceridemic subjects with and without diabetes to cultured skin fibroblasts was significantly decreased to 74 +/- 19% and 78 +/- 14% of that seen with LDL from normolipidemic nondiabetic subjects and diabetic normolipidemic controls (100 +/- 0%, 101 +/- 25%; P less than 0.005). Unlabeled LDL from hypertriglyceridemic subjects with and without diabetes failed to suppress LDL receptor activity and sterol synthesis from 14C-acetate as efficiently as unlabeled LDL from healthy subjects. The ability of LDL from hypertriglyceridemic subjects, whether diabetic or not, to suppress LDL binding was inversely related to the ratio of triglyceride to protein in LDL (r = 0.71, P less than 0.01) and showed a positive correlation with the LDL cholesterol/protein ratio (0.69, P less than 0.01). Thus, LDL from patients with hypertriglyceridemia, with or without coexistent diabetes, shows impaired binding to LDL receptors and less ability to downregulate LDL receptor activity and sterol synthesis than does LDL from normolipidemic diabetic and nondiabetic subjects. These findings suggest that factors associated with hypertriglyceridemia rather than with diabetes result in altered metabolism of LDL in these disorders.

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether.

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.

Phorbol esters inhibit the binding of low-density lipoproteins (LDL) to U-937 monocytelike cells.

The present study demonstrates that U-937 monocytelike human cells possess specific LDL receptors. 125I-LDL binds at 4 degrees C on the cell surface. The bound molecules are releasable by heparin. The reaction requires Ca2+ and the binding sites are sensitive to proteolysis. Unlabeled LDL compete with 125I-LDL, whereas HDL are ineffective. At 37 degrees C, LDL are internalized and degraded by a chloroquine-sensitive pathway. Tumor-promoting phorbol esters inhibit the binding of 125I-LDL to its receptor on U-937 cells. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, inhibition is 50% at 5 X 10(-9) M of TPA. After removal of phorbol esters, treated cells recover their 125I-LDL-binding activity in 60 min. The inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the number of available LDL receptors rather than a decrease in receptor affinity.

Factors controlling the release from human blood polymorphonuclear cells in vitro of a proteolytic activity directed against apolipoprotein A-II.

Human high-density lipoprotein class-3 (HDL3) was incubated with freshly isolated blood polymorphonuclear leukocytes (PMN) at 37 and 4 degrees C. At both temperatures the release of proteolytic activity (PA) causing the specific hydrolysis of apo-A-II was dependent on the concentration of HDL3 in the medium. At 37 degrees C, the efflux of PA was linear and no saturation was reached up to an HDL3 protein concentration in the medium of 800 micrograms/ml. In turn, at 4 degrees C, maximal PA release was reached at a concentration below 600 micrograms/ml of HDL3 protein/ml in the medium. Canine HDL, which contains apo-A-I, but not apo-A-II, was as effective as human HDL3 in promoting the release of PA from PMN. This property was also exhibited by egg lecithin/cholesterol vesicles containing apo-A-I. At 4 degrees C, there was no strict correlation between efflux of PA affected by HDL3 and specific binding of 125I-apo-A-I (HDL3). In competitive binding experiments, a 50-fold excess of unlabeled HDL3 prevented more than 90% of the binding of 125I-apo-A-I (HDL3) to PMN, whereas an excess of unlabeled low-density lipoprotein exhibited no effect. When human HDL3 was incubated with PMN at 4 or 37 degrees C and then subjected to ultracentrifugation at d 1.21 g/ml, most of the PA that was initially associated with this lipoprotein was recovered in the bottom of the tube. By gel filtration, both PA and HDL3 were in the same peak in a low ionic strength buffer, but were dissociated from each other by a high-salt solution (d 1.21 g/ml). We conclude that both naturally occurring HDLs and apo-A-I-stabilized lipid vesicles favor the release from PMN of an enzymatic activity which cleaves human apo-A-II. This release appears to be dependent both on the interaction of the cells with the lipoprotein ligand and on the lipoprotein surface area acting as the acceptor for the enzyme, probably through electrostatic forces.

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4°C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (< 50 pg protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 μM) and colchicine (1 μM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.

Degradation of lipoproteins by human monocyte-derived macrophages. Evidence for two distinct processes for the degradation of abnormal very-low-density lipoprotein from subjects with type III hyperlipidaemia.

Human blood monocyte-derived macrophages that had been cultured in medium containing human serum for 7 days degraded the abnormal very-low-density lipoproteins (VLDL) from the plasma of subjects with type III hyperlipoproteinaemia by two distinct saturable processes. One process was stimulated when cells from normal subjects were preincubated with lipoprotein-free medium, was inhibited by excess unlabelled low-density lipoproteins (LDL) and was absent from cells from subjects with homozygous familial hypercholesterolaemia; on these criteria it was identified as an LDL-receptor-dependent process. Degradation by the second process was of equal magnitude in both cell types and was unaffected by excess unlabelled LDL or acetylated LDL. The activity of this process was reduced when the cells were preincubated in lipoprotein-free medium. The abnormal VLDL from the plasma of cholesterol-fed rabbits were also degraded by this process, which was similar to that in mouse peritoneal macrophages mediated by the receptor for VLDL of beta-electrophoretic mobility [Goldstein, Ho, Brown, Innerarity & Mahley (1980) J. Biol. Chem. 255, 1839-1848].

In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells.

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.

Regulation of low density lipoprotein receptors by plasma lipoproteins from patients with abetalipoproteinemia.

Despite an absence of low density lipoproteins (LDLs) and chylomicron remnants from plasma, the rates of cholesterol synthesis or the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased. These observations suggest that other lipoprotein particles present in the plasma of patients with abetalipoproteinemia may regulate LDL receptor activity and the rates of cellular cholesterol synthesis in this disorder. In the present report we have studied the effects of lipoprotein fractions from the plasma of normal subjects, patients with abetalipoproteinemia, and a patient with dysbetalipoproteinemia on the binding, internalization, and degradation of 125I-labeled LDL (125I-LDL) by cultured human fibroblasts. LDL from normal subjects or the high density lipoprotein fraction HDL2 from the plasma of patients with abetalipoproteinemia effectively down-regulated LDL receptor activity (greater than 50% inhibition at 20 micrograms of protein per ml). HDL2 from the plasma of patients with abetalipoproteinemia also effectively reduced the binding, internalization, and degradation of 125I-LDL by cultured human fibroblasts. 125I-HDL2 from the plasma of patients with abetalipoproteinemia was bound, internalized, and degraded by cultured human fibroblasts; this process was competitively inhibited by unlabeled normal LDL or HDL2 from abetalipoproteinemic plasma and was 1/6th to 1/8th times as high when 125I-HDL2 was incubated with fibroblasts from a patient with receptor-negative homozygous familial hypercholesterolemia. We conclude that lipoproteins present in the HDL2 fraction of plasma from patients with abetalipoproteinemia (which are relatively rich in apoprotein E) are effective regulators of LDL receptor activity in normal human fibroblasts. These in vitro findings may explain why the in vivo rates of cholesterol synthesis and the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased.

Hydrocortisone decreases the internalization of low density lipoprotein in cultured human fibroblasts and arterial smooth muscle cells

The effect of hydrocortisone on the cellular low density lipoprotein (LDL) pathway was studied in cultured human skin fibroblasts and arterial smooth muscle cells. Hydrocortisone decreased both uptake and degradation of 125I-LDL, LDL binding, measured at 4 C, was not affected by the hormone. Physiological concentrations of hydrocortisone (4 . 1 X 10(-8) mol/l) resulted in a 30% reduction of LDL uptake and degradation which could not be accounted for by an effect of the hormone on macromolecular synthesis, cell protein or cell number. To test whether the decrease in uptake and degradation of 125I-LDL was due to reduced internalization, the effect of hydrocortisone on the internalization of prebound LDL was determined and found to be decreased. Also, preincubation with unlabelled LDL in the presence of hydrocortisone resulted in less down regulation of LDL receptor activity than when no hydrocortisone was present. An effect of the hormone on bulk phase endocytosis has been excluded, since hydrocortisone did not affect either LDL degradation by receptor negative cells or endocytosis of 14C-sucrose by normal skin fibroblasts. Thus, hydrocortisone impairs LDL catabolism by decreasing the internalization of LDL normally bound to its cell surface receptor. These results may be relevant to the pathogenesis of atherosclerosis in conditions associated with reduced cellular LDL catabolism.

Ultrastructural visualization of low-density lipoproteins during receptor binding and cellular endocytosis

Human fibroblasts possess surface receptors which have a high affinity for low-density lipoproteins (LDL). However, previous studies have not provided direct ultrastructural visualization of LDL bound to the receptor. To permit direct observation of unlabeled LDL during receptor binding and cellular endocytosis, we examined several fixative regimens which employ lipophilic stains. Staining with tannic acid, an oxidized form of ruthenium red, or potassium ferrocyanide imparted sufficient contrast to individual molecules of LDL to permit high-resolution electron microscopy of receptor binding and endocytosis. The LDL molecule was observed in immediate contact with the receptor and the coated vesicle, indicating that receptor-ligand binding occurs by short-range interactions.

Foam cells in explants of atherosclerotic rabbit aortas have receptors for beta-very low density lipoproteins and modified low density lipoproteins.

Beta-migrating very low density lipoproteins (beta--VLDL) and chemically modified low density lipoproteins (LDL), labeled with the fluorescent probe 3,3'-dioctadecylindocarbocyanine (Dil), were used to determine whether foam cells from atherosclerotic lesions have lipoprotein receptors similar to those of macrophages. The advantage of the Dil probe is that it produces a brilliant fluorescence due to its retention by the cells. When cells grown from intimal-medial explants of atherosclerotic rabbit aortas were incubated with Dil-labeled beta-VLDL, both smooth muscle cells and foam cells became fluorescent. However, when large amounts of unlabeled LDL were added to compete with the beta-VLDL for binding to the LDL receptors, Dil-labeled beta-VLDL binding to smooth muscle cells was blocked, and only the foam cells became highly fluorescent, due to the presence of specific receptors for beta-VLDL on those cells. The foam cells in the explants also became fluorescent when incubated with Dil-labeled acetoacetylated (AcAc) LDL. The same fluorescently labeled foam cells avidly bound IgG-coated erythrocytes. It was possible to inhibit the binding of the Dil-labeled AcAc LDL to aortic foam cells by the addition of fucoidin, a known inhibitor of modified LDL binding to macrophages. Foam cells that became fluorescent were similar to cells grown from the explants that contained an abundance of Oil red O positive lipid droplets and demonstrated high acid lipase activity. These results suggest that macrophages were the precursors of the foam cells derived from explants of atherosclerotic rabbit aortas. One difference we noted between these foam cells and macrophages, however, was that foam cells from the explants retained their cholesteryl esters under the same culture conditions that resulted in the loss of lipid from macrophages loaded with lipoproteins in vitro. The existence of receptors for beta-VLDL on aortic foam cells supports the contention that dietary cholesterol-induced beta-VLDL may be atherogenic lipoproteins in vivo.

Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure

Human plasma low density lipoproteins (LDL) are composed of approximately 25% apoproteins and 75% lipids (w/w). Immunochemical properties of LDL were studied using monoclonal antibodies. BALB/c mice were immunized with LDL and the spleen cells from these mice were then fused with a non-immunoglobulin secreting myeloma cell line (F0). The clones producing desirable antibodies were selected to study the antigenic properties of LDL by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. First, it was found that the maximal binding of 125I-labeled LDL to polyvinyl chloride microtiter dishes was not temperature dependent. The binding affinity was high with a Ka value of approximately 1.9 × 10 10M −1 while the monoclonal antibodies possessed an affinity to LDL of 5 × 10 8M −1 which was 2 orders less than the affinity of LDL to the dishes. The former binding, once established, was irreversible as judged by a subsequent incubation with an excess of unlabeled LDL. The latter binding could be displaced by unlabeled LDL. Therefore, the ELISA technique offered a satisfactory approach to study the interaction between LDL and monoclonal antibodies. Removal of lipids from bound LDL by organic extraction resulted in a 50% loss of immunoreactivity, suggesting that the lipids of LDL are important in maintaining the antigenic structure of LDL. Since the apoprotein of LDL also constitutes approximately 40% of the mass (w/w) of very low density lipoproteins (VLDL), the immunoreactivity of VLDL assessed by LDL-monoclonal antibodies was also carried out. Removal of triglycerides from VLDL by lipoprotein lipase resulted in a substantial loss of immunoreactivity as determined by radioimmunoassay. These findings are consistent with the concept that lipids play a role in maintaining the integrity of the antigenic structure of LDL.

Characterization of specific low-density lipoprotein binding sites in human term placental microvillous membranes

Purified microvillous membranes prepared from normal term human placenta were studied for their ability to bind specifically low-density lipoprotein (LDL). Electron microscopic examination of the membrane preparations revealed essentially microvilli-like structures, and the enzyme analyses a 14–17-fold enrichment in the membrane markers 5'-nucleotidase and alkaline phosphatase. The binding of [ 125I]LDL was dependent on time, temperature, pH and protein concentration; it was saturable with a low capacity (130.4 ± 22.2 ng/mg of membrane protein) and presented a high affinity (apparent K a 6.12 ± 1.32 μg protein per ml). These high-affinity binding sites were specific for LDL (high-density lipoprotein induced less competition than unlabelled LDL) and were sensitive to pronase digestion. Unlike the binding of LDL to other tissues, the [ 125I]LDL binding to microvillous membranes did not require divalent cations. The presence of specific LDL receptors on the placental microvillous membranes, located at the effective site of exchange between the maternal blood and the placental tissue, supports the concept that human placenta utilizes LDL-cholesterol for its progesterone synthesis.