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The purpose of the present study was to define the effect of low density lipoprotein (LDL) on progesterone (P) synthesis and on intracellular cholesterol content using monolayer cultured human luteal cells. When cultured in TC199 containing 10% lipoprotein poor serum (LPPS), P production decreased as culture time passed. In the presence of hCG, it also decreased, but slowly. In the presence of LDL, P production was kept at a higher level. Intracellular cholesterol content in luteal cells was measured by gas chromatography. The cholesterol content of luteal cells in a 4 day LPPS culture was higher than that in a 10 day LPPS culture. The cholesterol content in a 10 day LPPS culture with LDL was higher than that in a 10 day LPPS culture. The cholesterol content in a 10 day LPPS culture with hCG had a tendency to be lower than that in a 10 day LPPS culture, but there was no statistical difference (p less than 0.1). It was concluded that LDL was utilized in supplying intracellular cholesterol to luteal cells as a substrate for P synthesis. And in a state of LDL depletion in the medium, the luteal cells produced a small amount of P utilizing intracellular cholesterol. It was estimated from the results of the present study that hCG enhanced utilization of intracellular cholesterol.

The efficiency of supplying cholesterol by the LDL endocytic pathway of lymphoblastic T CEM cells was compared when incubated in the presence of either fetal calf serum (FCS) or lipoprotein-depleted fetal calf serum (LDFCS). In the presence of FCS, there were 8600 +/- 2000 LDL receptors/cell with a Kd of (2.2 +/- 0.8).10(-8) M and a receptor cycling time of about 7 min; about 90% of the internalized LDL was degraded. LDL degradation produced 98% of total cellular cholesterol and only 2% came from endogenous synthesis. The absence of LDL in the culture medium of lymphoblastic CEM cells deeply modified certain metabolic and structural characteristics of the cells. Their cholesterol content decreased; the total number of LDL receptors increased 6-fold, whereas their affinity for the ligand decreased by the same factor (Kd = (1.2 +/- 0.2).10(-7) M); the receptor cycling time increased 3-fold. Finally, LDL degraded by cholesterol-depleted CEM cells amounted to about 40% of that degraded by untreated CEM cells.

Most mammalian cells, such as fibroblasts, continuously internalize part of their surface membrane by endocytosis, and then later return it to the cell surface. This cyclical process is initiated by coated pits in the plasma membrane. These pits collect specific receptors plus lipid for internalization, but exclude other proteins. On a motile cell, the sites of endocytosis (randomly located on the cell) and those of membrane return (located at the front of the cell) are not coincident. This causes a bulk flow of lipid plus receptors in the plasma membrane, away from the front of the cell. Large objects on the cell surface are swept to the rear of the cell by this flow, a process called capping. Cells may use this polarized endocytic cycle to move.

We investigated lipoprotein metabolism in 14 patients with recessive X-linked ichthyosis (RXLI), a metabolic disease characterized by scaly skin, corneal opacity and steroid sulfatase deficiency. Plasma total cholesterol (TC) levels ranged from normal to slightly low (mean +/- SD: 156 +/- 28 mg/dl). Four patients showed a mild or moderate elevation of plasma triglyceride (TG) levels ranging from 150 to 365 mg/dl. The apoprotein B (apo B) to TC ratio was higher than in normal controls (0.63 +/- 0.11 vs. 0.52 +/- 0.07, P less than 0.01), while plasma apoB levels were within the normal range (99 +/- 17 mg/dl). Polyacrylamide gel electrophoretic mobility of low-density lipoprotein (LDL) was markedly increased in all patients, and further analyses showed that this finding was not due to a change in the particle size of the LDL but to an increased content of cholesterol sulfate (1.0-2.3% of the LDL-cholesterol content). In addition to the alteration of electrophoretic mobility, marked changes in the lipid and apoprotein compositions of the LDL fraction were observed; cholesterol ester content in LDL (LDL-CE) was significantly lower than that of control subjects (37 +/- 4% vs. 41 +/- 2% of total lipids, P less than 0.01), while the triglyceride content (LDL-TG) and apo B to cholesterol ratios in LDL were significantly higher than those of controls (18 +/- 7 vs. 10 +/- 2, P less than 0.001; 1.21 +/- 0.19 vs. 0.73 +/- 0.05, P less than 0.001, respectively). This anionized LDL, in which cholesterol sulfate was increased, was shown to bind to the LDL receptor of fibroblasts to much the same extent as normal LDL. In conclusion, the increase in cholesterol sulfate in LDL fraction not only alters the electrophoretic moiety but also the relative contents of apoB, cholesterol, and triglyceride in the lipoprotein. It does not change the affinity of LDL for the LDL receptor.

We studied the effects of beta-migrating very low density lipoprotein (beta-VLDL) on the vascular responses of isolated thoracic aortic preparations taken from normal and hypercholesterolemic rabbits. The endothelium-dependent relaxation induced by acetylcholine or adenosine triphosphate (ATP) was attenuated in the arteries from hypercholesterolemic rabbits that were fed a cholesterol-rich diet for 12 weeks. In these aortas, the lesional circumference of the atherosclerotic plaques (fatty streaks) was only 12.18 +/- 1.98%. The relaxation induced by the Ca2+ ionophore A23187 or nitroglycerin was not altered. Preincubation with beta-VLDL significantly inhibited the relaxation due to acetylcholine, ATP, or A23187, especially in the aortas of hypercholesterolemic rabbits. However, beta-VLDL did not alter the response to nitroglycerin. Preincubation with high density lipoprotein had no significant effect on vessel relaxation. These results indicated that endothelium-dependent relaxation was already inhibited in the early stages of atherosclerosis, and that the atherogenic lipoprotein, beta-VLDL, further inhibited endothelium-dependent relaxation in atherosclerotic aortas. It may be that beta-VLDL also plays a role in determining the level of vascular tonus in atherosclerosis.

The contribution of Escherichia coli hemolysin (ECH) to bacterial virulence has been considered mainly in context with its hemolytic properties. We here report that this prevalent bacterial cytolysin is the most potent leukocidin known to date. Very low concentrations (approximately 1 ng/ml) of ECH evoke membrane permeability defects in PMN (2-10 x 10(6) cells/ml) leading to an efflux of cellular ATP and influx of propidium iodide. The attacked cells do not appear to repair the membrane lesions. Human serum albumin, high density and low density lipoprotein, and IgG together protect erythrocytes and platelets against attack by even high doses (5-25 micrograms/ml) of ECH. In contrast, PMN are still permeabilized by ECH at low doses (50-250 ng/ml) in the presence of these plasma inactivators. Thus, PMN become preferred targets for attack by ECH in human blood and protein-rich body fluids. Kinetic studies demonstrate that membrane permeabilization is a rapid process, ATP-release commencing within seconds after application of toxin to leukocytes. It is estimated that membrane permeabilization ensues upon binding of approximately 300 molecules ECH/PMN. This process is paralleled by granule exocytosis, and by loss of phagocytic killing capacity of the cells. The recognition that ECH directly counteracts a major immune defence mechanism of the human organism through its attack on granulocytes under physiological conditions sheds new light on its possible role and potential importance as a virulence factor of E. coli.

A glycoprotein molecule discovered in pituitary glands of experimental animals is thought to be the precursor molecule for the pituitary peptides ACTH and beta-lipotropin, molecules themselves known to contain the amino acid sequences of several smaller peptides subsequently isolated. Evidence now exists to suggest the enzymatic cleavage of ACTH to alpha-MSH and corticotropic-like intermediate lobe peptide (CLIP), pituitary peptides with effects upon the fetal pituitary gland. Beta-lipotropin is probable the prohormone for the peptides beta-MSH, gamma-lipotropin, methionine-enkephalin, and beta-endorphin. Beta-MSH may enchance the known physiologic effects of mammalian central nervous system transmitters, while the enkephalins and beta-endorphin have been shown to exhibit opioid analgesic properties as well as effects upon behavior, temperature regulation, and the release of growth hormone and prolactin. Homologies among their amino acid sequences and evidence for prohormone activity in ACTH, beta-lipotropin, and the putative ACTH-beta lipotropin precursor suggest the possibility of the presence of a previously unsuspected interrelationship in the synthesis and release of these various pituitary peptides.

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Many human atherosclerotic lesions, showing no evidence of fissure or ulceration, contain a large amount of fibrin which may be in the form of mural thrombus on the intact surface of the plaque, in layers within the fibrous cap, in the lipid-rich centre, or diffusely distributed throughout the plaque. Small mural thrombi are invaded by SMCs and collagen is deposited in patterns closely resembling the early proliferative gelatinous lesions. In experimental animals, thrombi are converted into lesions with all the characteristics of fibrous plaques, and in saphenous-vein bypass grafts, fibrin deposition is the main cause of wall thickening and occlusion. There seems little doubt that fibrin deposition can both initiate atherogenesis and contribute to the growth of plaques. Epidemiological studies indicate that increased levels of fibrinogen and clotting activity are associated with accelerated atherosclerosis, and although blood fibrinolytic activity has given inconsistent results, in arterial intima both fibrinolytic activity and plasminogen concentration are decreased in cardiovascular disease. Fibrin may stimulate cell proliferation by providing a scaffold along which cells migrate, and by binding fibronectin, which stimulates cell migration and adhesion. Fibrin degradation products, which are present in the intima, may stimulate mitogenesis and collagen synthesis, attract leukocytes, and alter endothelial permeability and vascular tone. In the advanced plaque fibrin may be involved in the tight binding of LDL and accumulation of lipid. Thus there is extensive evidence that enhanced blood coagulation is a risk factor not only for thrombotic occlusion, but also for atherogenesis. Enhanced blood coagulation frequently coexists with hyperlipidaemia and, together, these may have a synergistic effect on atherogenesis.

A special in-depth substudy was conducted on 388 children from a total biracial (black-white) population, who were stratified on levels of serum beta- and pre-beta-lipoprotein cholesterol to explore factors associated with lipoprotein levels in childhood. Biochemical parameters on venous blood samples were obtained both on fasting subjects and after an abbreviated glucose tolerance test, along with selected anthropometric measures like height, weight, and skinfolds. Biochemical and anthropometric relationships were minimal for children with elevated beta-lipoprotein cholesterol and low pre-beta-lipoprotein cholesterol. On the other hand, children with higher levels of pre-beta-lipoprotein cholesterol, with or without elevated beta-lipoprotein cholesterol, showed associations with fatness and slightly higher levels of glucose and insulin, with other biochemical parameters considered within normal levels. These differences noted among free-living children with different levels of serum lipoproteins provide clues to mechanisms involved in the early natural history of coronary artery disease.

Interactions between vascular endothelium and low density lipoprotein (LDL) have been implicated in the development of atherosclerosis. The effect of normal and oxidized LDL (Ox-LDL) on prostaglandin release by cultured adult human saphenous vein endothelial cells was investigated. Ox-LDL induced a rapid release of prostacyclin (PGI2) to levels which were several-fold higher than those observed with control LDL. PGI2 release was concentration-dependent and was biphasic, with a first peak occurring within 30 minutes (followed by a decrease), and a second peak occurring after several hours of incubation. PGI2 production was inhibited by lipoprotein-depleted serum and by indomethacin, an antagonist of cyclooxygenase activity. These cells produced mainly PGF2 alpha, with some PGE2 and PGI2 when stimulated by the ionophore A23187 at confluency. However, among these prostanoids, mainly PGI2 was produced in response to Ox-LDL. The data indicate that Ox-LDL induces the production of PGI2 by human vascular endothelial cells. Since Ox-LDL is cytotoxic, this phenomenon may be a manifestation of an early response to injury.

The plasma lipoproteins are the primary means of transport of cholesterol among tissues. In particular, the apo B-containing lipoproteins (VLDL, IDL and LDL) are important for the delivery of cholesterol from the liver to peripheral tissues, while HDL appear to mediate the reverse process of movement of cholesterol from tissues back to the liver. Both of these transport processes are necessary for efficient whole body cholesterol homeostasis, because the liver is the major site of both the production and excretion of cholesterol. However, deviations from a proper balance of transport of cholesterol, either increases in LDL levels or decreases in HDL cholesterol flux, may result in accumulation of cholesterol in extrahepatic tissues. Increased risk of atherosclerosis and CHD may be associated with elevation in the number of LDL particles, increase or decrease in LDL particle size, or changes in the composition of plasma LDL. These modifications of plasma LDL may be brought about following perturbation of one of several aspects of LDL metabolism. These include decreased LDL receptor activity, increased VLDL production and cholesterol enrichment of the liver-derived VLDL. The events in the arterial wall that make some LDL particles apparently atherogenic are not well understood. In the case of nonhuman primates, large-size LDL are associated with an increased risk of CHD. One characteristic of these LDL is that their core lipids are rich in saturated cholesteryl esters and their transition temperatures are frequently above body temperature. The liquid crystalline cholesteryl ester cores of such LDL may modulate the conformation of apo B on the surface and thereby affect the interaction of these LDL with cellular receptors or connective tissue matrix proteoglycans. It is likely, though, that changes in LDL particle number, LDL particle size and LDL particle composition may each contribute to progression of atherosclerosis. The presumed metabolic events that make HDL protective against atherosclerosis have been termed reverse cholesterol transport, and suggest that small HDL that are deficient in free cholesterol acquire this lipid from cell membranes. The HDL cholesterol is esterified by LCAT in the circulation, forming large HDL that can then deliver the cholesteryl ester to the liver by both direct and indirect means. In most circumstances, it is assumed that an increase in plasma HDL cholesterol concentration reflects an increase in the rate at which HDL is removing cholesterol from tissues and, consequently, a decrease in atherosclerosis.(ABSTRACT TRUNCATED AT 400 WORDS)

A study was made of the in vitro effects of the abnormal serum lipoproteins associated with liver disease on the activity of the enzyme lecithin:cholesterol acyltransferase. At lipoprotein concentrations equivalent to those found in hepatic disease sera, the results indicate that: (1) LP-X levels greater than 2.5 mg/ml produced total inhibition of enzyme activity. (2) LP-X levels remained constant even up to 36 h incubation, despite active cholesterol esterification in the presence of LP-X concentrations less than 2.5 mg/ml. In addition, the specific activity of radiolabelled LP-X, and its electrophoretic properties remained unchanged after incubation showing that the molecule remained intact. (3) Low density lipoproteins other than LP-X stimulated the enzymes activity, but this effect was overcome by LP-X. (4) Additional concentrations of high density lipoproteins also produced enhancement of enzyme activity, but at higher levels inhibition was seen. LP-X prevented the enhancement of lecithin:cholesterol acyltransferase activity. (5) Small samples of pure LP-X, obtained with the minimum of physical manipulation, showed a complete absence of cholesterol ester and triacylglycerol from the molecule. The implications of these results are discussed, particularly in relation to other reports which have presented evidence that LP-X is a substrate for lecithin:cholesterol acyltransferase.

Low-density lipoproteins (LDL) have been shown to have a number of effects on the function of various cell types. To appreciate whether the in vitro effects of LDL have in vivo relevance, it is necessary to demonstrate that the biologic action described can be accounted for by native LDL and not by an alteration in the molecule or an addition to the preparation occurring during isolation. EDTA is frequently added to LDL during preparation to prevent oxidation. The effect of EDTA dialysis on LDL-mediated inhibition of lymphocyte responses was therefore examined. LDL alone did not inhibit mitogen-induced initial lymphocyte activation but rather suppressed lymphocyte DNA synthesis and subsequent proliferation in a transferrin-reversible manner. LDL dialysed with EDTA also inhibited lymphocyte responsiveness but the inhibition was not reversed by transferrin. Further experiments demonstrated that after dialysis EDTA in the LDL accounted for the change in its inhibitory effects. EDTA did not alter the lipoprotein but itself inhibited lymphocyte responses by chelating zinc necessary for DNA synthesis. These data indicate that LDL preparations may exhibit at least two separate effects on lymphocyte function. LDL is directly suppressive, while small amounts of contaminating EDTA can additionally be suppressive by chelating zinc. Thus, EDTA present in LDL preparations can alter their apparent biologic effects.

It has been proposed that an endogenously synthesized oxysterol mediates the regulation of cholesterol biosynthesis by low density lipoprotein in cultured mammalian cells. Studies in this report demonstrate that under conditions in which squalene conversion to sterols is blocked either by inhibition of squalene cyclization or lanosterol demethylation, or both, low density lipoprotein regulates 3-hydroxy-3-methylglutaryl coenzyme A reductase normally. These observations rule out the hypotheses that either an endogenously synthesized oxygenated cholesterol biosynthetic intermediate or epoxysterol is required to mediate the inhibition of this enzyme by low density lipoprotein.

Human serum high density lipoproteins (HDL), low density lipoproteins (LDL) and very low density lipoproteins (VLDL) were isolated and tested for their ability to inhibit Sindbis virus infectivity, hemagglutination and hemolysis. VLDL and LDL produced a strong reduction on both viral infectivity on Vero cell monolayers and attachment and fusion with erythrocytes, whereas HDL appeared to be only a weak inhibitor. Lipid and protein components were extracted from each class of lipoproteins to identify the molecules responsible for the inhibiting activity. Only the lipid moiety was found to inhibit Sindbis virus biological activities. Among the individual lipid components of lipoproteins, neutral lipids (cholesterol, oleic acid and palmitic acid) and negatively charged phospholipids (phosphatidylserine and phosphatidylinositol) and glycolipids (GM 3 ganglioside and cerebroside sulphate) were able to neutralize the virus suggesting that either hydrophobic or electrostatic interactions are involved in the inhibition.

Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol.

The influence of various oestrogens during unopposed replacement therapy on circulating androgens and cortisol was studied in 65 post-menopausal women. As dose dependent decrease in dehydroepiandrosterone sulphate (DHAS) was found. Ethinyloestradiol (0.05 mg daily) already gave a significant decrease after 1 mth of treatment. The decline following 17 beta-oestradiol (2 mg) and oestrone sulphate (2.5 mg) was less pronounced. Oestriol (6 mg daily) had no effect. Ethinyloestradiol also increased the levels of total cortisol and testosterone, probable because of serum protein induction, while 17 beta-oestradiol had no significant effect. Serum levels of androstenedione remained unchanged during therapy.

In man and in experimental animals, elevations in plasma cholesterol lead to premature atherosclerosis. It is likely that the cholesterol-rich low density lipoprotein (LDL) plays a keyrole in atherogenesis. LDL accumulates in patients with familial hypercholesterolemia (FH), a genetic syndrome associated with a defective LDL receptor in parenchymal cells and premature atherosclerosis. Monocytic/macrophage-like cells invading the vessel wall are becoming enriched with cholesteryl ester and concentrating in the early atherosclerotic lesion. Modification of LDL stimulates the uptake of cholesterol by macrophages in vitro leading to the conversion of the cells to lipid laden foam cells. Because of this phenomenon recent investigations were focussing on the lipid metabolism of macrophages. In vitro studies have demonstrated that macrophages have specific cell surface receptors for modified forms of LDL. It was suggested that these scavenger receptors could mediate foam cell formation in vivo, too. In vivo analysis by sequential scintiscans revealed that the liver is accumulating most actively modified LDL, possibly acting as a sieve for atherogenic lipoproteins. The putative liver receptor for modified (= atherogenic) LDL was characterized as a membrane protein of 220 000 to 250 000 D by ligand blotting.

Oxidized LDL (o-LDL) is toxic to a variety of cultured cells. Preliminary results suggested that susceptibility is enhanced by cell proliferation. As a step toward determining the mechanism of cytotoxicity, we chose to identify the cell cycle phase(s) during which exposure of cultured human fibroblasts to o-LDL leads to death. Cytochalasin B, which blocks cell migration and proliferation, and irradiation, which prevents mitosis but not migration, both blocked cytotoxicity. Colchicine, which arrests cells in mitosis but does not inhibit DNA synthesis, did not block cytotoxicity. Treatment of cells with hydroxyurea, which blocks cells prior to S phase, prevented cell death. Addition of o-LDL to cells immediately after S phase allowed mitosis without death. The above results coupled with results using cells synchronized by three different means indicate that cell death is selective for proliferating cells and occurs after exposure to o-LDL during S phase. Understanding the mechanism of o-LDL-induced death may have implications for tissue damage in vivo in the numerous instances of pathology in which oxidized lipoproteins or lipids are present.