The murine macrophage apoB-48 receptor gene (Apob-48r): homology to the human receptor

Previously we cloned the human macrophage apolipoprotein B-48 receptor (ApoB-48R) and documented its expression in human atherosclerotic foam cells (1). Now we have identified and characterized the murine macrophage apob-48r cDNA gene sequence and its chromosomal location. The cDNA (3,615 bp) -deduced amino acid (aa) sequence (942 aa) is approximately 45% identical to the human macrophage APOB-48R, but not to other known gene families. The murine Apob-48r gene, like the human APOB-48R gene, consists of four exons interrupted by three small introns and is syntenically located on chromosome 7. Functionally significant conserved domains include an N-terminal hydrophobic domain, a glycosaminoglycan attachment site, an N-glycosylation site, and an ExxxLL internalization motif C-terminal to the putative internal transmembrane domain. Two conserved coiled-coil domains are likely involved in the spontaneous homodimerization that generates the active dimeric ligand binding species (mouse, approximately 190 kDa; human, approximately 200 kDa). Transfection of the murine apoB-48R into Chinese hamster ovary cells (CHOs) confers apoB-48R function: rapid, high-affinity, specific uptake of known triglyceride-rich lipoprotein ligands of the apoB-48R and, of note, uptake of the cholesteryl ester-rich apoB-48-containing very low density lipoproteins that accumulate in atherosclerosis-prone apoE-deficient mice. Uptake of these ligands by murine apoB-48R-transfected CHOs causes saturable, visible cellular triglyceride and cholesterol accumulation in vitro that resemble foam cells of atherosclerotic lesions. In aggregate, the data presented here and that previously published suggest that the apoE-independent murine apoB-48R pathway may contribute to the spontaneous development of atherosclerotic lesions rich in macrophage-derived foam cells observed in apoE-deficient mice, a murine model of human atherosclerosis.     

Overexpression of SR-BI by adenoviral vector reverses the fibrateinduced hypercholesterolemia of apolipoprotein E-deficient mice

The hypercholesterolemia characteristic of apolipoprotein (apoE)-deficient mice fed on a regular chow diet is caused by the abnormal accumulation of apoB-48-carrying remnants of chylomicrons and very low density lipoproteins in the plasma. Treatment of apoE-deficient mice with ciprofibrate or other peroxisome proliferator-activated receptor alpha agonists severely aggravates their hypercholesterolemia by interfering with one or more mechanisms of remnant removal from the circulation that do not require mediation by apoE (Fu, T., Kashireddy, P., and Borensztajn, J. (2003) Biochem. J. 373, 941-947). In the present investigation we report that ciprofibrate treatment causes the down-regulation of hepatic scavenger receptor class B, type I (SR-BI) protein expression in the livers of apoE-deficient mice. On cessation of the treatment SR-BI expression returns to its pretreatment levels, coinciding with a reversal of the hypercholesterolemia to base-line concentrations. Restoration of SR-BI expression in ciprofibrate-treated apoE-deficient mice by recombinant adenoviral gene transfer abolishes the ciprofibrate-induced over accumulation of apoB-48-carrying remnants in the plasma. We also report that remnants isolated from the plasma of ciprofibrate-treated apoE-deficient mice bind to murine SR-BI expressed in stably transfected cultured cells. These observations suggest that, in addition to its well established role as high density lipoprotein receptor, SR-BI can also function as a remnant receptor responsible for the clearance of remnants from the circulation of apoE-deficient mice.     

Interaction of plasma-derived lipid transfer protein with macrophages in culture

This study investigates the ability of human plasma-derived lipid transfer protein to facilitate lipid transfer to and from intact viable cells in culture. Mouse peritoneal macrophages or J774 macrophages were preincubated with acetylated low density lipoprotein and [3H]oleate/albumin to promote the intracellular synthesis and accumulation of cholesteryl [3H]oleate and 3H-labeled triglyceride. The addition of partially purified lipid transfer protein to cultures of lipid-loaded macrophages resulted in a time and concentration-dependent transfer of radiolabeled cholesteryl ester and triglyceride from macrophages to the medium. At 48 hr, lipid transfer protein facilitated the net transfer of 16 and 11% of cellular cholesteryl ester and triglyceride radioactivity, respectively, to the medium; transfer in the absence of the lipid transfer protein was less than 2%. The transfer of cholesteryl ester radioactivity was accompanied by a similar decrease in cellular cholesteryl ester mass indicating a net transfer event. Lipid transfer from cells was not dependent on the presence of a lipoprotein acceptor in the medium; however, low and high density lipoproteins present at 200 micrograms cholesterol/ml did significantly stimulate the transfer protein-facilitated efflux of these lipids. Lipid transfer protein did not appear capable of transferring radiolabeled lipid from low density or high density lipoprotein to macrophages. Radiolabeled cholesteryl ester and triglyceride transferred from cells to the medium by lipid transfer protein were associated with large molecular weight (greater than 2 x 10(6)) components in the medium with an average density greater than 1.21 g/ml; these lipids were not associated with lipid transfer protein itself. However, these radiolabeled lipids were readily incorporated into low or high density lipoproteins when these lipoproteins were added to the medium either during or after its incubation with cells. It is concluded that lipid transfer protein can facilitate the net efflux of cholesteryl esters from intact, living macrophages. These studies suggest a novel and potentially antiatherogenic role for lipid transfer protein.     

Aggregated LDL and lipid dispersions induce lysosomal cholesteryl ester accumulation in macrophage foam cells

Macrophage foam cells in atherosclerotic lesions accumulate substantial cholesterol stores within large, swollen lysosomes. Previous studies with mildly oxidized low density lipoprotein (OxLDL)-treated THP-1 macrophages suggest an initial buildup of free cholesterol (FC), followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis and a subsequent lysosomal accumulation of unhydrolyzed lipoprotein CE. We examined whether other potential sources of cholesterol found within atherosclerotic lesions could also induce similar lysosomal accumulation. Biochemical analysis combined with microscopic analysis showed that treatment of THP-1 macrophages with aggregated low density lipoprotein (AggLDL) or CE-rich lipid dispersions (DISP) produced a similar lysosomal accumulation of both FC and CE. Co-treatment with an ACAT inhibitor, CP113,818, confirmed that the CE accumulation was primarily the result of the inhibition of lysosomal CE hydrolysis. The rate of unhydrolyzed CE buildup was more rapid with DISP than with AggLDL. However, with both treatments, FC appeared to accumulate in lysosomes before the inhibition in hydrolysis and CE accumulation, a sequence shared with mildly OxLDL. Thus, lysosomal accumulation of FC and CE can be attributable to more general mechanisms than just the inhibition of hydrolysis by oxidized lipids.     

Simvastatin causes the formation of cholesterol-rich remnants in mice lacking apoE

Mice that lack apolipoprotein E (apoE) display a severe hypercholesterolemia, caused by the accumulation of apolipoprotein B-48 (apoB-48)-carrying remnants of chylomicrons and very-low-density lipoproteins in the plasma. Statins are potent inhibitors of cholesterol synthesis that, when administered to mice lacking apoE, cause paradoxical further increases in plasma cholesterol levels. In the present study, we examined the mechanisms responsible for this phenomenon. ApoE-deficient mice fed a chow diet containing simvastatin developed, as anticipated, an enhanced increase in plasma cholesterol and a decrease in plasma triglycerides. Fractionation of the plasma lipoproteins by FPLC revealed that the lipid changes were confined to the lipoprotein remnants. Western blot analysis of the remnants from the untreated and simvastatin-treated mice showed no differences in their apoB-48 content, indicating that both groups of animals accumulated similar numbers of remnant particles in the plasma. Following the injection of Triton WR-1339, the simvastatin-treated mice accumulated in the plasma significantly more cholesterol and significantly less triglycerides than the untreated animals. These results indicate that the enhanced hypercholesterolemia observed in apoE-deficient mice treated with simvastatin is not the result of an increased number of remnant particles in circulation but is caused by synthesis and secretion into the plasma of lipoproteins that are enriched in cholesterol and depleted of triglycerides.     

Deficiency in apolipoprotein E has a protective effect on diet-induced nonalcoholic fatty liver disease in mice

Apolipoprotein E (apoE) mediates the efficient catabolism of the chylomicron remnants very low-density lipoprotein and low-density lipoprotein from the circulation, and the de novo biogenesis of high-density lipoprotein. Lipid-bound apoE is the natural ligand for the low-density lipoprotein receptor (LDLr), LDLr-related protein 1 and other scavenger receptors. Recently, we have established that deficiency in apoE renders mice resistant to diet-induced obesity. In the light of these well-documented properties of apoE, we sought to investigate its role in the development of diet-induced nonalcoholic fatty liver disease (NAFLD). apoE-deficient, LDLr-deficient and control C57BL/6 mice were fed a western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5 kcal·g(-)) for 24 weeks and their sensitivity to NAFLD was assessed by histological and biochemical methods. apoE-deficient mice were less sensitive than control C57BL/6 mice to diet-induced NAFLD. In an attempt to identify the molecular basis for this phenomenon, biochemical and kinetic analyses revealed that apoE-deficient mice displayed a significantly delayed post-prandial triglyceride clearance from their plasma. In contrast with apoE-deficient mice, LDLr-deficient mice fed a western-type diet for 24 weeks developed significant accumulation of hepatic triglycerides and NAFLD, suggesting that apoE-mediated hepatic triglyceride accumulation in mice is independent of LDLr. Our findings suggest a new role of apoE as a key peripheral contributor to hepatic lipid homeostasis and the development of diet-induced NAFLD.     

Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes

The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets.These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells.     

Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver

ApoE-deficient mice on low fat diet show hepatic triglyceride accumulation and a reduced very low density lipoprotein (VLDL) triglyceride production rate. To establish the role of apoE in the regulation of hepatic VLDL production, the human APOE3 gene was introduced into apoE-deficient mice by cross-breeding with APOE3 transgenics (APOE3/apoe-/- mice) or by adenoviral transduction. APOE3 was expressed in the liver and, to a lesser extent, in brain, spleen, and lung of transgenic APOE3/apoe-/- mice similar to endogenous apoe. Plasma cholesterol levels in APOE/apoe-/- mice (3.4 +/- 0.5 mM) were reduced when compared with apoe-/- mice (12.6 +/- 1.4 mM) but still elevated when compared with wild type control values (1.9 +/- 0.1 mM). Hepatic triglyceride accumulation in apoE-deficient mice was completely reversed by introduction of the APOE3 transgene. The in vivo hepatic VLDL-triglyceride production rate was reduced to 36% of control values in apoE-deficient mice but normalized in APOE3/apoe-/- mice. Hepatic secretion of apoB was not affected in either of the strains. Secretion of (3)H-labeled triglycerides synthesized from [(3)H]glycerol by cultured hepatocytes from apoE-deficient mice was four times lower than by APOE3/apoe-/- or control hepatocytes. The average size of secreted VLDL particles produced by cultured apoE-deficient hepatocytes was significantly reduced when compared with those of APOE3/apoe-/- and wild type mice. Hepatic expression of human APOE3 cDNA via adenovirus-mediated gene transfer in apoE-deficient mice resulted in a reduction of plasma cholesterol depending on plasma apoE3 levels. The in vivo VLDL-triglyceride production rate in these mice was increased up to 500% compared with LacZ-injected controls and correlated with the amount of apoE3 per particle. These findings indicate a regulatory role of apoE in hepatic VLDL-triglyceride secretion, independent from its role in lipoprotein clearance.     

Carboxyl ester lipase expression in macrophages increases cholesteryl ester accumulation and promotes atherosclerosis

Carboxyl ester lipase (CEL, also called cholesterol esterase or bile salt-dependent lipase) is a lipolytic enzyme capable of hydrolyzing cholesteryl esters, triacylglycerols, and phospholipids in a trihydroxy bile salt-dependent manner but hydrolyzes ceramides and lysophospholipids via bile salt-independent mechanisms. Although CEL is synthesized predominantly in the pancreas, a low level of CEL expression was reported in human macrophages. This study used transgenic mice with macrophage CEL expression at levels comparable with that observed in human macrophages to explore the functional role and physiological significance of macrophage CEL expression. Peritoneal macrophages from CEL transgenic mice displayed a 4-fold increase in [(3)H]oleate incorporation into cholesteryl [(3)H]oleate compared with CEL-negative macrophages when the cells were incubated under basal conditions in vitro. When challenged with acetylated low density lipoprotein, cholesteryl ester accumulation was 2.5-fold higher in macrophages expressing the CEL transgene. The differences in cholesteryl ester accumulation were attributed to the lower levels of ceramide and lysophosphatidylcholine in CEL-expressing cells than in CEL-negative cells. CEL transgenic mice bred to an atherosclerosis susceptible apoE(-/-) background displayed an approximate 4-fold higher atherosclerotic lesion area than apoE(-/-) mice without the CEL transgene when both were fed a high fat/cholesterol diet. Plasma level of the atherogenic lysophosphatidylcholine was lower in the CEL transgenic mice, but plasma cholesterol level and lipoprotein profile were similar between the two groups. These studies documented that CEL expression in macrophages is pro-atherogenic and that the mechanism is because of its hydrolysis of ceramide and lysophosphatidylcholine in promoting cholesterol esterification and decreasing cholesterol efflux.     

Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma

In a previous study we demonstrated that highly purified lipid-transfer protein facilitated the transfer of triglyceride, cholesteryl ester, and phosphatidylcholine between plasma lipoproteins. It remained unclear, however, whether these lipids were transferred by independent sites on the lipid-transfer protein. To address this point, we have studied the protein-mediated transfer of triglyceride, cholesteryl ester, and phosphatidylcholine as a function of the concentration and lipid composition of donor and acceptor lipoproteins. Lipoproteins labeled in vitro, reconstituted lipoproteins of defined lipid composition, and phosphatidylcholine liposomes with or without triglyceride and/or cholesteryl ester have been used to investigate the inter-relationships of lipids transferred by the lipid-transfer protein. In studies of initial (less than or equal to 10-13%) transfer, we found that, although absolute transfer rates were affected, the ratio of cholesteryl ester to triglyceride transferred was independent of donor and acceptor lipoprotein concentrations and acceptor lipoprotein lipid composition. With reconstituted lipoproteins as donor, we demonstrated that this ratio was linearly related to the ratio of cholesteryl ester to triglyceride in the donor particle; the sum of triglyceride and cholesteryl ester transferred remained constant and independent of the lipid composition of the donor. Experiments with intact lipoproteins labeled in vitro and with small unilamellar vesicles in the presence and absence of p-chloromercuriphenylsulfonate, confirmed the interdependence of triglyceride and cholesteryl ester transfer. In contrast, under all assay conditions, no correlation was found between the amount of phosphatidylcholine transferred and the transfer of triglyceride and/or cholesteryl ester. We conclude that triglyceride and cholesteryl ester compete for transfer and that the extent of transfer for each lipid is determined by its relative concentration in the donor particle, whereas phosphatidylcholine transfer is independent of triglyceride and cholesteryl ester transfer. The data also strongly support the conclusion that lipid transfer protein promotes both the exchange and net transfer of triglyceride and cholesteryl ester and that the net transfer process proceeds by a reciprocal exchange of triglyceride and cholesteryl ester without net transfer of core lipid between lipoproteins.     

Markedly increased secretion of VLDL triglycerides induced by gene transfer of apolipoprotein E isoforms in apoE-deficient mice

Apolipoprotein E (apoE) plays a key role in the receptor-mediated uptake of lipoproteins by the liver and therefore in regulating plasma levels of lipoproteins. ApoE may also facilitate hepatic secretion of very low density lipoprotein (VLDL) triglyceride (TG). We directly tested the hypothesis that reconstitution of hepatic apoE expression in adult apoE-deficient mice by gene transfer would acutely enhance VLDL-TG production and directly compared the three major human apoE isoforms using this approach. Second generation recombinant adenoviruses encoding the three major isoforms of human apoE (E2, E3, and E4) or a control virus were injected intravenously into apoE-deficient mice, resulting in acute expression of the apoE isoforms in the liver. Despite the expected decreases in total and VLDL cholesterol levels, apoE expression was associated with increased total and VLDL triglyceride levels (E2 > E4 > E3). The increase in TG levels significantly correlated with plasma apoE concentrations. In order to determine whether acute apoE expression influenced the rate of VLDL-TG production, additional experiments were performed. Three days after injection of adenoviruses, Triton WR1339 was injected to block lipolysis of TG-rich lipoproteins and VLDL-TG production rates were determined. Mice injected with control adenovirus had a mean VLDL-TG production rate of 74 +/- 7 micromol/h/kg. In contrast, VLDL-TG production rates in apoE-expressing mice were 363 +/- 162 micromol/h/kg, 286 +/- 175 micromol/h/kg, and 300 +/- 84 micromol/h/kg for apoE2, apoE3, and apoE4, respectively. The VLDL-TG production rates in apoE-expressing mice were all significantly greater than in control mice but were not significantly different from each other. In summary, acute expression of all three human apoE isoforms in livers of apoE-deficient mice markedly increased VLDL-TG production to a similar degree, consistent with the concept that apoE plays an important role in facilitating hepatic VLDL-TG production in an isoform-independent manner.     

Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone

In macrophages, the accumulation of cholesteryl esters synthesized by the activated acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) results in the foam cell formation, a hallmark of early atherosclerotic lesions. In this study, with the treatment of a glucocorticoid hormone dexamethasone (Dex), lipid staining results clearly showed the large accumulation of lipid droplets containing cholesteryl esters in THP-1-derived macrophages exposed to lower concentration of the oxidized low-density lipoprotein (ox-LDL). More notably, when treated together with specific anti-ACAT inhibitors, the abundant cholesteryl ester accumulation was markedly diminished in THP-1-derived macrophages, confirming that ACAT is the key enzyme responsible for intracellular cholesteryl ester synthesis. RT-PCR and Western blot results indicated that Dex caused up-regulation of human ACAT1 expression at both the mRNA and protein levels in THP-1 and THP-l-derived macrophages. The luciferase activity assay demonstrated that Dex could enhance the activity of human ACAT1 gene P1 promoter, a major factor leading to the ACAT1 activation, in a cell-specific manner. Further experimental evidences showed that a glucocorticoid response element (GRE) located within human ACAT1 gene P1 promoter to response to the elevation of human ACAT1 gene expression by Dex could be functionally bound with glucocorticoid receptor (GR) proteins. These data supported the hypothesis that the clinical treatment with Dex, which increased the incidence of atherosclerosis, may in part due to enhancing the ACAT1 expression to promote the accumulation of cholesteryl esters during the macrophage-derived foam cell formation, an early stage of atherosclerosis.     

Stable overexpression of human macrophage cholesteryl ester hydrolase results in enhanced free cholesterol efflux from human THP1 macrophages

Reduction of the lipid burden of atherosclerotic lesion-associated macrophage foam cells is a logical strategy to reduce the plaque volume. Since extracellular cholesterol acceptor-mediated cholesterol efflux is the only recognized mechanism of cholesterol removal from foam cells and this process is rate limited at the level of intracellular cholesterol ester hydrolysis, a reaction catalyzed by neutral cholesteryl ester hydrolase (CEH), we examined the hypothesis that CEH overexpression in the human macrophage monocyte/macrophage cell line THP1 results in increased cholesterol efflux, as well as decreased cellular cholesterol ester accumulation. We generated THP1-CEH cells with stable integration of human macrophage CEH cDNA driven by the cytomegalovirus promoter. Compared with wild-type THP1 cells (THP1-WT), THP1-CEH cells showed increased CEH mRNA expression and increased CEH activity. Efflux of free or unesterified cholesterol by acetylated LDL-loaded THP1-CEH cells to ApoA-I by an ABCA1-dependent pathway or to HDL by an ABCG1-dependent pathway was significantly higher than that in THP1-WT cells. In addition, THP1-CEH cells accumulated significantly lower amount of esterified cholesterol. CEH overexpression, therefore, not only enhances cholesterol efflux but also reduces cellular accumulation of cholesteryl esters. Taken together, these data provide evidence for evaluating CEH expression in human macrophages as a potential target for attenuation of foam cell formation and regression of atherosclerotic plaques. lipoproteins; lipid burden; foam cells     

Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-gamma.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-gamma (IFN-gamma), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG -VLDL. Incubation of cells with HTG -VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Pre-incubation of cells with IFN-gamma (50 U/ml) prior to incubation with HTG -VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-gamma increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-gamma did not enhance the uptake of type III (apoE2/E2) HTG -VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-gamma-enhanced HTG -VLDL uptake was dependent on LPL and ACAT activities. IFN-gamma significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled alpha2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-gamma-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-gamma may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-gamma-stimulated CE accumulation in the presence of HTG -VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-gamma-enhanced uptake of both HTG -VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.     

Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages

Oxidatively modified low density lipoproteins (Ox-LDL) may be involved in determining the formation of foam cells by inducing cellular cholesteryl ester accumulation. We studied the effect of copper oxidized LDL (Ox-LDL) on cholesterol accumulation and esterification in murine macrophages. Ox-LDL (44 micrograms/ml of lipoprotein cholesterol) increased the total cholesterol content of the cells from 29 to 69 micrograms/mg cell protein. Free cholesterol accounted for 85% of this increase. Acetyl LDL (Ac-LDL) (38 micrograms/ml of lipoprotein cholesterol), raised total cellular cholesterol content to a similar extent (76 micrograms/mg cell protein), however only 25% of the accumulated cholesterol was unesterified. When ACAT activity was determined after incubation of J774 cell with Ox- or Ac-LDL, Ox-LDL were 12 times less effective than Ac-LDL in stimulating cholesteryl ester formation. This was not due to an inhibition of ACAT by Ox-LDL since these lipoproteins failed to inhibit pre activated enzyme in cholesteryl ester-loaded macrophages. The uptake of 125I-Ox-LDL: was 175% that of 125I-Ac-LDL, while degradation was only 20%. All together these data suggest an altered intracellular processing of Ox-LDL, which may be responsible for free cholesterol accumulation.     

Manzamine A,a marine-derived alkaloid,inhibits accumulation of cholesterol ester in macrophages and suppresses hyperlipidemia and atherosclerosis in vivo

The formation of foam cells in macrophages plays an essential role in the progression of early atherosclerotic lesions and therefore its prevention is considered to be a promising target for the treatment of atherosclerosis. We found that an extract of the marine sponge Acanthostrongylophora ingens inhibited the foam cell formation induced by acetylated low-density lipoprotein (AcLDL) in human monocyte-derived macrophages, as measured based on the accumulation of cholesterol ester (CE). Bioassay-guided purification of inhibitors from the extract afforded manzamines. Manzamine A was the most potent inhibitor of foam cell formation, and also suppressed CE formation in Chinese hamster ovary cells overexpressing acyl-CoA:cholesterol acyl-transferase (ACAT)-1 or ACAT-2. In addition, manzamine A inhibited ACAT activity. Next, we orally administered manzamine A to apolipoprotein E (apoE)-deficient mice for 80 days, and found that total cholesterol, free cholesterol, LDL-cholesterol, and triglyceride levels in serum were significantly reduced and the area of atherosclerotic lesions in the aortic sinus was also substantially diminished. These findings clearly suggest that manzamine A suppresses hyperlipidemia and atherosclerosis in apoE-deficient mice by inhibiting ACAT and is therefore a promising lead compound in the prevention or treatment of atherosclerosis. Although manzamine A has been reported to show several biological activities, this is the first report of a suppressive effect of manzamine A on atherosclerosis in vivo.     

Characterization of the binding site on thioglycolate-stimulated mouse peritoneal macrophages that mediates the uptake of very low density lipoproteins

Isolated mouse peritoneal macrophages that had been stimulated with thioglycolate were shown to take up and degrade normal human 125I-very low density lipoproteins (VLDL). Uptake occurred via a specific cell surface receptor which was shown to be 1) temperature-dependent, 2) calcium-dependent, and 3) susceptible to proteolytic digestion. The receptor-mediated uptake and degradation of VLDL markedly stimulated the synthesis and accumulation of triglyceride and cholesteryl ester within macrophages. The degradation of the protein and lipid portions of VLDL occurred within lysosomes. Competition studies showed that the binding site for VLDL was different from the receptor for normal low density lipoproteins or for acetylated low density lipoproteins but that there was cross competition with beta-VLDL. In addition, positive charges appeared to play an important role in the recognition of VLDL by their receptors since polyamines were able to markedly inhibit VLDL binding, degradation, and lipid accumulation while negatively charged compounds were without effects. These studies indicate that 1) stimulated mouse peritoneal macrophages possess specific receptors which recognize normal human VLDL and 2) the receptor-mediated uptake of VLDL results in the accumulation of triglyceride and cholesteryl ester within macrophages.     

Effects of cellular cholesterol loading on macrophage foam cell lysosome acidification

Macrophages incubated with mildly oxidized low density lipoprotein (OxLDL), aggregated low density lipoprotein (AggLDL), or cholesteryl ester-rich lipid dispersions (DISPs) accumulate cholesterol in lysosomes followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis. The variety of cholesterol-containing particles producing inhibition of hydrolysis suggests that inhibition may relate to general changes in lysosomes. Lysosome pH is a key mediator of activity and thus is a potential mechanism for lipid-induced inhibition. We investigated the effects of cholesterol accumulation on THP-1 macrophage lysosome pH. Treatment with OxLDL, AggLDL, and DISPs resulted in inhibition of the lysosome's ability to maintain an active pH and concomitant decreases in CE hydrolysis. Consistent with an overall disruption of lysosome function, exposure to OxLDL or AggLDL reduced lysosomal apolipoprotein B degradation. The lysosomal cholesterol sequestration and inactivation are not observed in cholesterol-equivalent cells loaded using acetylated low density lipoprotein (AcLDL). However, AcLDL-derived cholesterol in the presence of progesterone (to block cholesterol egression from lysosomes) inhibited lysosome acidification. Lysosome inhibition was not attributable to a decrease in the overall levels of vacuolar ATPase. However, augmentation of membrane cholesterol in isolated lysosomes inhibited vacuolar ATPase-dependent pumping of H+ ions into lysosomes. These data indicate that lysosomal cholesterol accumulation alters lysosomes in ways that could exacerbate foam cell formation and influence atherosclerotic lesion development.     

The bridging function of hepatic lipase clears plasma cholesterol in LDL receptor-deficient "apoB-48-only" and "apoB-100-only" mice

Hepatic lipase clears plasma cholesterol by lipolytic and nonlipolytic processing of lipoproteins. We hypothesized that the nonlipolytic processing (known as the bridging function) clears cholesterol by removing apoB-48- and apoB-100-containing lipoproteins by whole particle uptake. To test our hypotheses, we expressed catalytically inactive human HL (ciHL) in LDL receptor deficient "apoB-48-only" and "apoB-100-only" mice. Expression of ciHL in "apoB-48-only" mice reduced cholesterol by reducing LDL-C (by 54%, 46 +/- 6 vs. 19 +/- 8 mg/dl, P < 0.001). ApoB-48 was similarly reduced (by 60%). The similar reductions in LDL-C and apoB-48 indicate cholesterol removal by whole particle uptake. Expression of ciHL in "apoB-100-only" mice reduced cholesterol by reducing IDL-C (by 37%, 61 +/- 19 vs. 38 +/- 12 mg/dl, P < 0.003). Apo-B100 was also reduced (by 27%). The contribution of nutritional influences was examined with a high-fat diet challenge in the "apoB-100-only" background. On the high fat diet, ciHL reduced IDL-C (by 30%, 355 +/- 72 vs. 257 +/- 64 mg/dl, P < 0.04) but did not reduce apoB-100. The reduction in IDL-C in excess of apoB-100 suggests removal either by selective cholesteryl ester uptake, or by selective removal of larger, cholesteryl ester-enriched particles. Our results demonstrate that the bridging function removes apoB-48- and apoB-100-containing lipoproteins by whole particle uptake and other mechanisms.