Uptake and degradation of low density lipoprotein by swine arterial smoot muscle cells with inhibition of cholesterol biosynthesis.

We have previously proposed on the basis of studies in hepatectomized animals that low density lipoproteins are degraded at a significant rate by peripheral tissues. To test the capacity of one peripheral cell type to catabolize low density lipoprotein, cultures of swine aortic smooth muscle cells were incubated with homologous 125I-labeled low density lipoprotein and uptake and degradation measured. Degradation of 125I-labeled low density lipoprotein to products soluble in trichloroacetic acid showed an initial lag period of 1--2 h after which the rate increased and remained linear for the following 15 h. Rates of degradation increased sharply with low density lipoprotein concentration over the lower range (from 0--25 mug protein/ml) and then more slowly up to the highest concentration tested, 300 mug protein/ml. Even at very low concentrations, 1 mug low density lipoprotein protein/ml (less than 10% of the plasma low density lipoprotein concentration), the in vitro degradation rate (per kg of smooth muscle cells) exceeded the in vivo degradation rate (per kg of total body weight). To the extent that smooth muscle cells are representative of other peripheral cells, the results support the proposal that peripheral degradation of low density lipoprotein apoprotein may be quantitatively important. The rate of incorporation of labeled acetate into sterols was suppressed in cells incubated with whole serum, low density and very low density lipoproteins, or suspensions of free cholesterol. In this respect, the results were similar to those observed in human skin fibroblasts studied concurrently. However, high density lipoprotein inhibited sterol synthesis by about 25% in swine smooth muscle cells while it had no effect in human skin fibroblasts.     

The removal of cholesterol from aortic smooth muscle cells in culture and Landschutz ascites cells by fractions of human high-density apolipoprotein.

Ascites cells were labeled by intraperitoneal injection of [3H]cholesterol and aortic smooth muscle cells by addition of [3H]cholesterol to the serum component of the culture medium. The release of cholesterol from cells into a serum-free medium supplemented with the various "acceptors" was studied using ascites cells in suspension and aortic smooth muscle cells in a multilayer culture. Unfractionated human high-density apolipoprotein was somewhat more effective in the removal of labeled cellular free cholesterol, in both cell types, than apolipoprotein derived from rat high-density lipoprotein. Following separation of human high-density apolipoprotein into four fractions by Sephadex chromatography, the effect of each fraction on the removal of cellular cholesterol from ascites cells was studied. The individual fractions had a lower capacity for cholesterol removal than the original unfractionated high-density apolipoprotein and the lowest activity was detected in Fraction II which comprised 75% of the total apolipoprotein. The effectiveness to remove cholesterol could be restored to all the fractions, as well as enhanced, by addition of sonicated suspensions of lecithin or sphingomyelin, which by themselves promoted a more limited removal of cellular cholesterol. Negatively stained preparations of mixtures of the four fractions and sonicated dispersion of lecithin were shown to consist of vesicles and discs of various sizes. Addition of the apolipoprotein fractions (especially Fractions II and IV) to sonicated dispersion of sphingomyelin resulted in a pronounced formation of discs which showed a high tendency towards stack formation. Mixtures of Fraction II and lecithin or sphingomyelin were effective in the release of cellular cholesterol from multilayers of aortic smooth muscle cells in culture. These results show the feasibility of net removal of cholesterol from cells which grow in a form resembling a tissue and thus provide a model to study the role of apolipoprotein-phospholipid mixtures in cholesterol removal from cells and tissues in vivo.     

Cholesterol metabolism in pigeon aortic smooth muscle cells lacking a functional low density lipoprotein receptor pathway

Cholesterol metabolism was examined in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons that have been shown to lack a functional LDL receptor pathway. In cells incubated in the presence of whole serum or low density lipoprotein (LDL) the rate of cholesterol synthesis from [1-14C]acetate or of HMG-CoA reductase activity was 20-100 times greater than for mammalian cells incubated under the same conditions. Unexpectedly, cholesterol synthesis decreased by nearly 50% after preincubation for 24 hr with lipoprotein-deficient serum (LPDS). This occurred without a change in cellular cholesterol content. Neither the high rate of cholesterol synthesis nor the effect of LPDS could be accounted for by differences in cell turnover or state of growth. Cholesterol added in ethanol was ineffective in altering cellular cholesterol synthesis or esterification even though a near doubling in cellular free cholesterol content occurred. Cholesterol synthesis and esterification were, however, able to be regulated with 25-OH cholesterol and mevalonolactone, as indicated by their ability to suppress cholesterol synthesis and to stimulate cholesterol esterification. In spite of the high rate of endogenous cholesterol synthesis, cellular cholesterol content was maintained at a constant level by the efficient efflux of the newly synthesized cholesterol from the cell. Unlike mammalian cells that require a cholesterol acceptor in the medium for efflux to occur, cholesterol efflux from pigeon cells occurred in the absence of a cholesterol acceptor. This suggests either that pigeon cells utilize a different mechanism for cholesterol efflux or that they produce their own cholesterol acceptor. As a result of a lack of a functional LDL receptor pathway, pigeon smooth muscle cells do not maintain cholesterol homeostasis through the controlled uptake of exogenous LDL cholesterol, as do mammalian cells. Rather, pigeon smooth muscle cells would appear to regulate cholesterol concentrations at the level of either cholesterol synthesis or efflux.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Effects of cholesterol sulfate on lipid metabolism in cultured human keratinocytes and fibroblasts

Effects of cholesterol sulfate on acetate incorporation into lipid fractions were examined in normal human fibroblast and keratinocyte cultures. Inhibition of sterologenesis in normal fibroblast cultures by cholesterol sulfate was less profound than that produced by either lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate also inhibited sterologenesis in low density lipoprotein receptor-deficient fibroblasts and inhibited both sterologenesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in keratinocytes. Cholesterol sulfate increased incorporation of acetate into fatty acid-containing lipids in preconfluent cultures of both cell types in lipoprotein-depleted media. Similar effects were not observed either in response to lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate had no effect on oleic acid incorporation into diglycerides, triglycerides, or phospholipid fractions; neither did it inhibit acid lipase activity; nor did it inhibit fatty acid oxidation, indicating that cholesterol sulfate does not inhibit catabolism of acyl lipids. Because cholesterol sulfate had similar effects on fatty acid metabolism in steroid sulfatase-deficient fibroblasts lines, desulfation to cholesterol is not a prerequisite. Cholesterol sulfate did not significantly affect incorporation of oleic acid into sterol esters in fibroblast cultures, but in contrast, inhibited sterol esterification in keratinocyte cultures. These data suggest a novel role for cholesterol sulfate as a modulator of cellular lipid biosynthesis.     

Lipoprotein and cholesterol metabolism in rabbit arterial endothelial cells in culture.

Like all other peripheral cells types thus far studied in culture, endothelial cells derived from the rabbit aorta bind, internalize and degrade low density lipoprotein (LDL) at a significant rate. At any given LDL concentration, the metabolism by rabbit endothelial cells was slower than that by fibroblasts or smooth muscle cells. Thus, longer incubations were required to achieve a net increment in cell cholesterol content or to suppress endogenous sterol synthesis; after 18-24 h incubation in the presence of LDL at 100 microgram LDL protein/ml inhibition was greater than 80% relative to the rate in cells incubated in the absence of lipoproteins. High density lipoproteins (HDL) were also taken up and degraded but did not inhibit sterol synthesis. Studies of LDL binding to the cell surface suggested the presence of at least two classes of binding sites; the high-affinity binding sites were fully saturated at very low LDL concentrations (about 5 microgram LDL protein/ml). However, the degree of inhibition of endogenous sterol synthesis increased progressively with increasing LDL concentrations from 5 to 100 microgram LDL/ml, suggesting that uptake from the low affinity sites in this cell line contributes to the suppression of endogenous sterol synthesis. The internalization and degradation of LDL also increased with concentrations as high as 700 microgram/ml. Thus, in vivo, where the cells are exposed to LDL concentrations far above that needed to saturate the high affinity sites, most of the LDL degradation would be attributable to LDL taken up from low affinity sites. As noted previously in swine arterial smooth muscle cells and in human skin fibroblasts, unlabeled HDL reduced the binding, internalization and degradation of labeled LDL. Cells incubated for 24 h in the presence of high concentrations of LDL alone showed a net increment in cell cholesterol content; the simultaneous presence of HDL in the medium significantly reduced this LDL-induced increment in cell cholesterol content. The possible relationship between LDL uptake and degradation by these cells in vitro is discussed in relationship to their transport function in vivo.     

Plasma membrane sphingomyelin and the regulation of HMG-CoA reductase activity and cholesterol biosynthesis in cell cultures

We have examined the mechanism of the inhibition of cholesterol synthesis in cells treated with exogenous sphingomyelinase. Treatment of rat intestinal epithelial cells (IEC-6), human skin fibroblasts (GM-43), and human hepatoma (HepG2) cells in culture with sphingomyelinase resulted in a concentration- and time-dependent inhibition of the activity of HMG-CoA reductase, a key regulatory enzyme in cholesterol biosynthesis. The following observations were obtained with IEC-6 cells. Free fatty acid synthesis or general cellular protein synthesis was unaffected by the addition of sphingomyelinase. Addition of sphingomyelinase to the in vitro reductase assay had no effect on activity, suggesting that an intact cell system is required for the action of sphingomyelinase. The products of sphingomyelin hydrolysis, e.g., ceramide and phosphocholine, had no effect on reductase activity. Sphingosine, a further product of ceramide metabolism, caused a stimulation of reductase activity. Examination of the incorporation of [3H]acetate into the nonsaponifiable lipid fractions in the presence of sphingomyelinase showed no changes in the percent distribution of radioactivity in the post-mevalonate intermediates of the cholesterol biosynthetic pathway, but there was increased radioactivity associated with the polar sterol fraction. Pretreatment of cells with ketoconazole, a known inhibitor of oxysterol formation, prevented the inhibition of reductase activity by sphingomyelinase and decreased the incorporation of [3H]acetate in the polar sterol fraction. Ketoconazole had no effect on exogenous sphingomyelinase activity in vitro in the presence or absence of cells. Endogenous sphingomyelinase activity was also unaffected by ketoconazole. Addition of inhibitors of endogenous sphingomyelinase activity, e.g., chlorpromazine, desipramine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), to the culture medium caused a dose-dependent stimulation of reductase activity. However, these agents had no effect on the inhibition of reductase activity by exogenous sphingomyelinase. Treatment of cells with small unilamellar vesicles of dioleyl phosphatidylcholine or high density lipoprotein3 resulted in increased efflux of cholesterol and stimulation of reductase activity. Under similar conditions, the inhibitory effect of exogenous sphingomyelinase on reductase activity was prevented by incubation with small unilamellar vesicles of phosphatidylcholine or high density lipoprotein. These results support the hypothesis that alteration of the ratio of sphingomyelin:cholesterol in the plasma membrane plays a modulatory role on the flow of membrane cholesterol to a site where it may be converted to a putative regulatory molecule, possibly an oxysterol.     

Lipoprotein-mediated efflux of radiolabeled cholesterol from cells does not indicate net removal of cellular cholesterol mass

The efflux of cholesterol from human skin fibroblasts was determined using radioisotope techniques and mass measurements. When the cells were labeled with [14C]- or [3H]-cholesterol and then incubated with very low density, low density, or high density lipoproteins or with serum, 20 to 30% of the label was released into the medium in 20 h. However, when the cellular cholesterol content was determined after incubation with various lipoproteins under identical conditions, only the heavier subfraction of high density lipoproteins (HDL3) caused a significant decrease in cellular cholesterol. This net removal of cholesterol can be observed in the cells without overloading them with cholesterol, by incubation with low density lipoproteins. Time studies indicated that at least 24 h of incubation is required to detect significant removal of cellular cholesterol. These experiments show that methods using the release of labeled cholesterol from cultured cells to determine net cholesterol removal mediated by high density lipoprotein, although currently used by many investigators, can lead to erroneous conclusions when employed without the measurement of cholesterol mass.     

Apolipoprotein-mediated cellular lipid release requires replenishment of sphingomyelin in a phosphatidylcholine-specific phospholipase C-dependent manner

When sphingomyelin is digested by sphingomyelinase in the plasma membrane of rat astrocytes, productions of sphingomyelin, diacylglycerol, and phosphatidylcholine are stimulated. D609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed these effects. Similarly, when apolipoprotein A-I removed cellular cholesterol, phosphatidylcholine, and sphingomyelin to generate high density lipoprotein, cholesterol synthesis from acetate subsequently increased, and sphingomyelin synthesis from acetate and serine also increased. D609 inhibited these effects again. D609 also inhibited the cholesterol removal by apoA-I not only from the astrocytes but also from BALB/3T3 and RAW264 cells. D609 decreased cholesterol synthesis, although D609 did not directly inhibit hydroxymethylglutaryl-CoA reductase. ApoA-I-stimulated translocation of newly synthesized cholesterol to cytosol was also decreased by D609. A diacylglycerol analog increased the apoA-I-mediated cholesterol release, whereas ceramide did not influence it. We concluded that removal of cellular sphingomyelin by apolipoproteins is replenished by transfer of phosphorylcholine from phosphatidylcholine to ceramide, and this reaction may limit the removal of cholesterol by apoA-I. This reaction also produces diacylglycerol that potentially triggers subsequent cellular signal cascades and regulates intracellular cholesterol trafficking.     

Suppression of cholesterol synthesis in cultured fibroblasts from a patient with homozygous familial hypercholesterolemia by her own low density lipoprotein density fraction A possible role of apolipoprotein E

The suppression of cellular cholesterol synthesis by low density lipoprotein (LDL) from a normal and from a homozygous familial hypercholesterolemic subject was measured on normal fibroblasts and on fibroblasts derived from the same homozygous familial hypercholesterolemic patient. On normal fibroblasts both LDL preparations (denisty 1.019 to 1.063 g/ml) exerted a similar suppression of cellular cholesterol synthesis. With the homozygous familial hypercholesterolemic fibroblasts homozygous hypercholesterolemic LDL suppressed the cholesterol synthesis to a much greater extent than did LDL from a normal subject. Analysis of lipid and protein composition of both LDL preparations showed that homozygous hypercholesterolemic LDL differs from normal LDL. In the homozygous hypercholesterolemic LDL preparation the ratio phosphatidylcholine to sphingomyelin is decreased, and even when taking a narrower density range (1.023 to 1.045 g/ml), apolipoprotein E is present. In this homozygous hypercholesterolemic LDL preparation (density range 1.023 to 1.045 g/ml) apolipoprotein E could be present as an integral LDL protein constituent or as an apolipoprotein of an HDL_c-like lipoprotein class with a floatation density similar to that of LDL. It is suggested that the presence of apolipoprotein E in the LDL density fraction from plasma from this homozygous familial hypercholesterolemic patient could offer an additional means for suppression of cellular cholesterol synthesis of this patient.     

Metabolism of cholesterol and phospholipids in cultured human vascular smooth muscle cells: differences between artery and vein-derived cells and the effect of oxygen partial pressure

Human smooth muscle (SM) cells derived from vena saphena magna, aorta abdominalis and arteria mamaria were grown in culture under 40 or 145 mmHg oxygen partial pressure (pO2) and their lipid metabolism studied. Esterification of the cellular [3H]cholesterol was higher by 2.5-fold in artery derived than in vein-derived cells and was slightly higher in cultures exposed to 145 mmHg than to 40 mmHg pO2. Cholesterol efflux in the presence of high density lipoprotein (HDL) in the incubation medium was higher in artery-derived than vein-derived cells. Apolipoprotein (apo) AI also supported cholesterol efflux to a higher extent in artery than in vein-derived cells. Cholesterol efflux in the presence of apo AI was accompanied by a decrease of 50% in cellular [3H]cholesteryl ester in both cell types. SM cultures exposed to [3H]choline incorporated about 90% of the radioactivity to phosphatidylcholine (PC) and 10% to sphingomyelin (SPM). During 5 days exposure to [3H]choline, 10 to 15% and 20 to 30% of the newly synthesized PC and SPM, respectively, were released by vein-derived cells into the incubation medium. The relative amount of SPM of the total radioactive phospholipids released by vein-derived cultures was significantly higher in cultures growing under 40 mmHg than 145 mmHg pO2 reaching a value of up to 33% of the radioactive phospholipids in the incubation medium. HDL was shown to serve as an acceptor for phospholipids released by both vein and artery-derived SM cells, while free apo AI supported phospholipid efflux in artery but not in vein-derived SM cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol-sphingomyelin interaction in membrane and apolipoprotein-mediated cellular cholesterol efflux

Helical apolipoproteins interact with cellular surface and generate high density lipoprotein (HDL) by removing phospholipid and cholesterol from cells. We have reported that the HDL is generated by this reaction with the fetal rat astrocytes and meningeal fibroblasts but cholesterol is poorly available to this reaction with the astrocytes (Ito et al. 1999. J. Neurochem. 72: 2362;-2369). Partial digestion of the membrane by extracellular sphingomyelinase increased the incorporation of cholesterol into thus-generated HDL from both types of cell. This increase was diminished by supplement of endogenous or exogenous sphingomyelin to the cells. The sphingomyelinase treatment decreased cholesterol in the membrane mainly in the detergent-resisting domain. The intracellular cholesterol used by acylCoA:cholesterol acyltransferase increased by the sphingomyelinase treatment in the absence of apoA-I, more remarkably in the fibroblast than in the astrocytes. ApoA-I suppressed this increase completely in the astrocytes, but only partially in the fibroblast. The effect of the sphingomyelin digestion was more prominent for the apolipoprotein-mediated reaction than the diffusion-mediated cellular cholesterol efflux. Thus, cholesterol molecules restricted by sphingomyelin in the domain of the plasma membrane appear to be primarily used for the HDL assembly upon the apolipoprotein;-cell interaction.     

Cholesterol efflux from cells to immunopurified subfractions of human high density lipoprotein: LP-AI and LP-AI/AII.

Using immunoaffinity chromatography, we separated human high density lipoprotein (HDL) into two subfractions: LP-AI, in which all particles contain apolipoprotein A-I (apoA-I) but no apoA-II, and LP-AI/AII, in which all particles contain both apoA-I and apoA-II. To compare LP-AI and LP-AI/AII as acceptors of cell cholesterol, the isolated subfractions were diluted to 50 micrograms phospholipid/ml, and then incubated with monolayer cultures of cells in which whole-cell and lysosomal cholesterol has been labeled with 14C and 3H, respectively. We used three cell types (Fu5AH rat hepatoma cells, normal human skin fibroblasts, and rabbit aortic smooth muscle cells). When these cells were prepared to contain normal physiological quantities of cholesterol (20-35 micrograms/mg protein), LP-AI and LP-AI/AII were nearly equally efficient in promoting efflux of both whole-cell and lysosomal cholesterol. For whole-cell cholesterol, the rate constants for efflux to LP-AI and LP-AI/AII were: 0.050/h and 0.053/h, respectively, with Fu5AH cells; 0.0063/h and 0.0074/h with GM3468 human skin fibroblasts; and 0.0076/h and 0.0079/h with rabbit aortic smooth muscle cells. When cholesterol in hepatoma cells or fibroblasts was elevated two- to threefold above normal, there was still not difference in efflux of whole-cell cholesterol to LP-AI and LP-AI/AII. In longterm incubations, the net depletion of cholesterol mass from cholesterol-enriched cells was either identical with the two HDL subfractions, or somewhat greater with LP-AI/AII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.     

Inhibition of human lymphocyte E-rosette formation by oxygenated sterols.

25-Hydroxycholesterol, 20 alpha-hydroxycholesterol, 7 alpha-hydroxycholesterol, and 5 alpha-hydroxy-6-ketocholestanol, when added to cultures of human lymphocytes in lipoprotein-depleted medium (LPDM) at a concentration of 2.5 x 10(-6) M, inhibit E-rosette formation with sheep red blood cells. 20 alpha-Hydroxycholesterol, 7 alpha-hydroxycholesterol, and 5 alpha-hydroxy-6-ketocholestanol are more potent inhibitors than 25-hydroxycholesterol. The inhibitory effect of 5 alpha-hydroxy-6-ketocholestanol on E-rosette formation appears after 15 min of exposure; with the other three compounds, an exposure time of 18 hr is necessary. The inhibitory effect of E-rosette formation can be abolished by addition of free cholesterol, low-density lipoprotein, or high-density lipoprotein to the LPDM or by incubation of the cells in normal AB serum, but not by the addition of mevalonic acid to the LPDM. These observations suggest that the capacity of oxygenated sterol compounds (OSC) to inhibit E-rosette formation is independent of their inhibitory effect on sterol synthesis. It is possible that OSC inhibit E-rosette formation as a consequence of their insertion into the lymphocyte membrane as cholesterol analogues.     

Apolipoprotein AIMilano. Disulfide-linked dimers increase high density lipoprotein stability and hinder particle interconversion in carrier plasma

The in vitro metabolism of high density lipoproteins (HDL) in carriers of the apolipoprotein AIMilano (apoAIM) mutant was investigated during incubation of whole plasma and isolated lipoprotein fractions. A reduced cholesterol esterification (16.5 versus 25.0% for controls) and a decreased exchange of lipids between HDL and lower density lipoproteins was observed during incubation (6 h at 37 degrees C) of AIM plasma. Control HDL3 were converted to larger, faster-floating HDL particles, whereas only a fraction of AIM HDL3 followed the same pathway. Incubations were also carried out by mixing HDL3 from controls and AIM carriers with a lipoprotein-depleted plasma fraction in the presence of triglyceride-rich particles isolated from Intralipid. AIM HDL3 again showed a reduced capacity for lipid exchange; some HDL3 particles followed a "normal" conversion to faster-floating, larger HDL, whereas the small AIM HDL3 were not modified, indicating that AIM HDL3 are a mixture of metabolically functional and nonfunctional particles. Following transformation of the apoAIM homo- and heterodimers into their normal counterparts, i.e. monomeric apoAI and -AII, by reduction and carboxamidomethylation of AIM HDL3, the modified HDL3 behave like control HDL3 during incubation with lipoprotein-depleted plasma and triglyceride-rich particles. The presence of AIM dimers is most likely responsible for the increased HDL3 stability in the AIM carriers, indicating that apolipoprotein composition plays a major role in HDL particle interconversion.     

Effects of sphingomyelin degradation on cholesterol mobilization and efflux to high-density lipoproteins in cultured fibroblasts

The hydrolysis of sphingomyelin from cellular plasma membranes imposes many consequences on cellular cholesterol homeostasis by causing a rapid and dramatic redistribution of plasma membrane cholesterol within the cells (Slotte, J.P. and Bierman, E.L. (1988) Biochem. J. 250, 653-658). The objective of this study was to examine the effects of an extracellular cholesterol acceptor on the directions of the sphingomyelinase-induced cholesterol flow in cultured fibroblasts. We have used HDL3 as a physiological acceptor for cholesterol, and measured the effects of sphingomyelin hydrolysis on efflux and endogenous esterification of cellular [3H]cholesterol. Treatment of cells with sphingomyelinase did induce a dramatically increased esterification of plasma-membrane-derived [3H]cholesterol. The presence of HDL3 in the medium (100 micrograms/ml) did not prevent or reduce the extent of the sphingomyelinase-induced cellular esterification of [3H]cholesterol. Degradation of cellular sphingomyelin (75% hydrolysis) also did not enhance the rate of [3H]cholesterol efflux from the plasma membranes to HDL3. In addition, we also observed that the degradation of sphingomyelin in the HDL3 particles (complete degradation) did not change the apparent rate of [3H]cholesterol transfer from HDL3 to the cells. These findings together indicate that hydrolysis of sphingomyelin did not markedly affect the rates of cholesterol surface transfer between HDL3 and cells. By whatever mechanism cholesterol is forced to be translocated from the plasma membranes subsequent to the degradation of sphingomyelin, it appears that the sterol flow is specifically directed towards the interior of the cells.     

Hyaluronan accumulation is elevated in cultures of low density lipoprotein receptor-deficient cells and is altered by manipulation of cell cholesterol content

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.     

Provision of cholesterol to lymphocytes by high density and low density lipoproteins. Requirement for low density lipoprotein receptors

The capacity of lipoprotein fractions to provide cholesterol necessary for human lymphocyte proliferation was examined. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. All lipoprotein fractions with the exception of high density lipoprotein subclass 3 were able to provide cholesterol for lymphocyte proliferation. Each of the lipoprotein subfractions capable of providing cholesterol was also able to regulate endogenous sterol synthesis in cultured human lymphocytes. Provision of cholesterol by lipoproteins required the interaction of apolipoprotein B or apolipoprotein E with specific receptors on normal lymphocytes. Apolipoprotein modification by acetylation or methylation, which markedly reduced the ability to regulate sterol biosynthesis, also diminished the capacity of lipoproteins to provide cholesterol. In addition, depletion of apolipoprotein B- and apolipoprotein E-containing particles from high density lipoprotein decreased its ability to suppress cholesterol synthesis and prevented it from providing cholesterol to proliferating lymphocytes. Monoclonal antibodies directed against the receptor-recognition sites on apolipoprotein B and apolipoprotein E were used to define the specific apolipoproteins required for the provision of cholesterol to lymphocytes by the various lipoprotein fractions. The antibody to apolipoprotein B inhibited cholesterol provision by both low density lipoprotein (LDL) and other lipoprotein fractions. The antibody to apolipoprotein E did not decrease provision of cholesterol by LDL but did inhibit the capacity of other fractions to provide cholesterol. In addition, a monoclonal antibody against the ligand binding site on the LDL receptor inhibited provision of cholesterol to normal lymphocytes by all lipoproteins. Finally, lymphocytes lacking LDL receptors were unable to obtain cholesterol from any lipoprotein fraction. These studies demonstrate that LDL receptor-mediated interaction with apolipoprotein B or apolipoprotein E is essential for the provision of cholesterol to normal human lymphocytes from all lipoprotein sources.     

Receptor-dependent uptake of human chylomicron remnants by cultured skin fibroblasts

Human chylomicrons were isolated from plasma from a subject with familial hypertriglyceridemia and converted to chylomicron remnants by incubation with postheparin plasma. The interaction of these apolipoprotein E-containing, cholesterol-rich human chylomicron remnants with cultured skin fibroblasts was studied. Chylomicron remnants were internalized by skin fibroblasts as a unit, mainly via the low density lipoprotein (LDL)-receptor pathway, resulting in increased cell cholesterol content. After entering the fibroblast, chylomicron remnants stimulated cholesterol esterification, suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and down-regulated LDL receptor activity similar to the action of LDL. As a function of increasing lipolysis, remnant particles were progressively more effectively taken up by skin fibroblasts, despite a decrease in the apolipoprotein E content per lipoprotein particle. Remnant particles produced after hydrolysis of 70 to 80% of chylomicron triglyceride increased cell cholesterol content to an amount nearly identical to that observed with LDL when the two lipoproteins were incubated at an equal cholesterol concentration. However, when incubated on the basis of equal particle number, chylomicron remnants were 2 to 3 times more effective than LDL in delivering cholesterol to the cells. These results suggest that chylomicron remnants play a role in the regulation of postabsorptive cholesterol homeostasis in nonhepatic cells, and possibly in the pathogenesis of atherosclerosis.