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I Tabas

Publications and source records attributed to I Tabas.

At least 55 records · Page 3Linked to original sources
PubMed1996

Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins.

Aggregation and retention of LDL in the arterial wall are key events in atherogenesis, but the mechanisms in vivo are not known. Previous work from our laboratories has shown that exposure of LDL to bacterial sphingomyelinase (SMase) in vitro leads to the formation of LDL aggregates that can be retained by extracellular matrix and that are able to stimulate macrophage foam cell formation. We now provide evidence that retained LDL is hydrolyzed by an arterial-wall SMase activity. First, we demonstrated that SMase-induced aggregation is caused by an increase in particle ceramide content, even in the presence of excess sphingomyelin (SM). This finding is compatible with previous data showing that lesional LDL is enriched in SM, though its ceramide content has not previously been reported. To address this critical compositional issue, the ceramide content of lesional LDL was assayed and, remarkably, found to be 10-50-fold enriched compared with plasma LDL ceramide. Furthermore, the ceramide was found exclusively in lesional LDL that was aggregated; unaggregated lesional LDL, which accounted for 20-25% of the lesional material, remained ceramide poor. When [3H]SM-LDL was incubated with strips of rabbit aorta ex vivo, a portion of the LDL was retained, and the [3H]SM of this portion, but not that of unretained LDL, was hydrolyzed to [3H]ceramide by a nonlysosomal arterial hydrolase. In summary, LDL retained in atherosclerotic lesions is acted upon by an arterial-wall SMase, which may participate in LDL aggregation and possibly other SMase-mediated processes during atherogenesis.

Animals↗
PubMed1996

Evidence that the initial up-regulation of phosphatidylcholine biosynthesis in free cholesterol-loaded macrophages is an adaptive response that prevents cholesterol-induced cellular necrosis. Proposed role of an eventual failure of this response in foam cell necrosis in advanced atherosclerosis.

Macrophages in atherosclerotic lesions accumulate free cholesterol (FC) as well as cholesteryl ester and appear to have high rates of phospholipid (PL) synthesis and increased PL mass. Previous short term (i.e. </=24 h) studies with cultured macrophages have shown that these cells respond to FC loading by up-regulating phosphatidylcholine biosynthesis. We propose that this response is adaptive by keeping the FC:PL ratio in the macrophages from reaching toxic levels. We further propose that one cause of macrophage necrosis, a prominent and important event in atherosclerosis, is an eventual decrease of this adaptive response. To explore these ideas, cultured macrophages were loaded with FC for up to 4 days and assayed for phosphatidylcholine biosynthesis, FC and PL mass, and cytotoxicity. For the first 24 h, cellular phosphatidylcholine biosynthesis and FC and PL mass increased 3-4-fold, and thus the FC:PL molar ratio was prevented from reaching very high levels; at this point, there were no overt signs of cytotoxicity. Over the next 24-48 h, however, phosphatidylcholine biosynthesis, and then phosphatidylcholine mass, began to decrease. Initially, the macrophages remained healthy and continued to accumulate FC, but eventually these macrophages, but not unloaded macrophages, became necrotic (swollen organelles and disrupted membranes). Lipoprotein dose studies indicated a close relationship between the onset of macrophage necrosis and the FC:PL ratio. To test further the causal nature of these relationships, cellular FC and PL mass were independently manipulated by using high density lipoprotein3 (HDL3) to decrease cellular FC and choline depletion to decrease cellular PC. As predicted by our hypotheses, HDL3 protected FC-loaded macrophages from necrosis, whereas choline depletion accelerated cytotoxic changes. These findings support the idea that the initial increase in phosphatidylcholine biosynthesis in FC-loaded macrophages is an adaptive response that prevents cholesterol-induced macrophage necrosis. We propose that an eventual failure of the PL response in foam cells may represent one cause of macrophage necrosis in advanced atherosclerotic lesions.

Arteriosclerosis↗
PubMed1996

Zn2+-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene.

Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. Although several mammalian sphingomyelinases have been identified and studied, one of these, an acidic Zn2+-stimulated sphingomyelinase (Zn-SMase) originally found in fetal bovine serum, has received little attention since its first and only report 7 years ago. We now show that Zn-SMase activity is secreted by human and murine macrophages, human skin fibroblasts, microglial cells, and several other cells in culture and is markedly up-regulated during differentiation of human monocytes to macrophages. Remarkably, peritoneal macrophages from mice in which the acid SMase gene had been disrupted by homologous recombination secreted no Zn-SMase activity, indicating that this enzyme and the intracellular lysosomal SMase, which is Zn-independent, arise from the same gene. Furthermore, skin fibroblasts from patients with types A and B Niemann-Pick disease, which are known to lack lysosomal SMase activity, also lack Zn-SMase activity in their conditioned media. Chinese hamster ovary cells stably transfected with a cDNA encoding lysosomal SMase massively overexpress both cellular lysosomal SMase and secreted Zn-SMase activities. Thus, Zn-SMase arises independently of alternative splicing, suggesting a post-translational process. In summary, a wide variety of cell types secrete Zn-SMase activity, which arises from the same gene as lysosomal SMase. This secreted enzyme may play roles in physiological and pathophysiological processes involving extracellular sphingomyelin hydrolysis.

Animals↗
PubMed1996

The distal pathway of lipoprotein-induced cholesterol esterification, but not sphingomyelinase-induced cholesterol esterification, is energy-dependent.

The stimulation of the intracellular cholesterol esterification pathway by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. The esterification pathway can also be stimulated by hydrolysis of cell-surface sphingomyelin by the enzyme sphingomyelinase (SMase). In both cases, intracellular cholesterol transport to the cholesterol esterifying enzyme, acyl-CoA:cholesterol O-acyltransferase (ACAT), is thought to be critical, although the mechanism of cholesterol transport is not known. In this report, we explore two fundamental properties of the cholesterol esterification pathway, namely its dependence on energy and the effect of other treatments that block membrane vesicle trafficking. After the atherogenic lipoprotein, beta-very low density lipoprotein (beta-VLDL), was internalized by macrophages and hydrolyzed in lysosomes, the cells were depleted of energy by treatment with sodium azide and 2-deoxyglucose or by permeabilization. Under these conditions, which allowed equal beta-VLDL-cholesteryl ester hydrolysis, cholesterol esterification was markedly decreased in the energy-depleted cells. This effect was not due to blockage of lysosomal cholesterol export. In the permeabilized cell system, energy repletion restored beta-VLDL-induced cholesterol esterification. Remarkably, stimulation of cholesterol esterification by SMase was not inhibited by energy depletion. Energy depletion also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification in Chinese hamster ovary cells. Similar experiments were carried out using N-ethylmaleimide, low potassium medium, or inhibitors of phosphatidylinositol 3-kinase, each of which blocks intracellular membrane vesicle trafficking. These treatments also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification. Finally, we show here that SMase treatment of cells leads to an increase in plasma membrane vesiculation that is relatively resistant to energy depletion. In summary, the stimulation of cholesterol esterification by lipoproteins, but not by SMase, is energy-dependent, N-ethylmaleimide-sensitive, and blocked by both low potassium and phosphatidylinositol 3-kinase inhibitors. The affected step or steps are distal to cholesterol export from lysosomes and not due to direct inhibition of the ACAT enzyme. Thus, the mechanisms involved in lipoprotein-induced versus SMase-induced cholesterol esterification are different, perhaps due to the involvement of energy-dependent vesicular cholesterol transport in the lipoprotein pathway and a novel, energy-independent vesicular transport mechanism in the SMase pathway.

Animals↗
PubMed1996

Interleukin 8 is induced by cholesterol loading of macrophages and expressed by macrophage foam cells in human atheroma.

In order to identify novel genes expressed in macrophage-derived foam cells, we used a multigene assay to examine the expression of genes in control versus cholesterol-loaded macrophages. We compared THP-1 macrophages incubated with or without acetylated LDL (acLDL) +/- acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor (compound 58035) for 20 h and assessed changes in mRNA of chemokines, growth factors, interleukins, and adhesion molecules. Among 49 genes examined, an increase in mRNA was observed only for interleukin 8 (IL-8) in THP-1 macrophages. Northern analysis confirmed a 3- to 4-fold increase of IL-8 mRNA and an enzyme-linked immunosorbent assay (ELISA) revealed a corresponding increase in IL-8 in conditioned medium. Oxidized LDL (oxLDL) also induced IL-8 mRNA, but native LDL had no effect. 58035 had a moderate effect on IL-8 induction by acLDL. AcLDL-induced IL-8 expression was concentration- and time-dependent. The time course of IL-8 induction paralleled that of cholesterol loading. MCP-1, a chemokine implicated in recruiting monocytes in atherogenesis, was also induced by acLDL. The induction of MCP-1, however, peaked at 1 h after addition of acLDL and returned to basal level by 20 h while IL-8 induction peaked at 8 h and was still 2-fold higher than basal level at 20 h. IL-8 induction was also observed in fresh human monocyte-derived macrophage cells treated with acLDL. Finally, immunohistochemistry and in situ hybridization studies using specimens of human coronary atheromas showed expression of IL-8 mRNA in a macrophage-rich area. We conclude that IL-8 is induced in macrophage foam cells as a response to cholesterol loading. The chemoattractant and/or mitogenic effects of IL-8 on neutrophils, T cells, smooth muscle, or vascular endothelial cells may contribute to the progression and complications of atherosclerosis.

Arteriosclerosis↗
PubMed1995

Stimulation of CTP:phosphocholine cytidylyltransferase by free cholesterol loading of macrophages involves signaling through protein dephosphorylation.

Free cholesterol-loaded macrophages in atheromata synthesize excess phosphatidylcholine (PC), which may be an important adaptive response to the excess free cholesterol (FC) load. We have recently shown that FC loading of macrophages leads to 2-4-fold increases in PC mass and biosynthesis and to the post-translational activation of the membrane-bound form of CTP:phosphocholine cytidylyltransferase (CT), a key enzyme in PC biosynthesis. Herein, we explore further the mechanism of CT activation in FC-loaded macrophages. First, enrichment of membranes from control macrophages with FC in vitro did not increase CT activity, and PC biosynthesis in vivo is up-regulated by FC loading even when CT and FC appear to be mostly in different intracellular sites. These data imply that FC activates membrane-bound CT by a signaling mechanism. That the proposed signaling mechanism involves structural changes in the CT protein was suggested by data showing that two different antibodies against synthetic CT peptides showed increased recognition of membrane-bound CT from FC-loaded cells despite no increase in CT protein. Since CT is phosphorylated, two-dimensional maps of peptides from 32P-labeled control and FC-loaded macrophages were compared: six peptide spots from membrane-bound CT, but none from soluble CT, were dephosphorylated in the FC-loaded cells. Furthermore, incubation of FC-loaded macrophages with the phosphatase inhibitor, calyculin A, blocked increases in both PC biosynthesis and antipeptide-antibody recognition of CT. Last, treatment of membranes from control macrophages with lambda phage protein phosphatase in vitro increased both CT activity (2-fold) and antipeptide-antibody recognition of CT; soluble CT activity and antibody recognition were not substantially affected by phosphatase treatment. In summary, FC loading of macrophages leads to the partial dephosphorylation of membrane-bound CT, and possibly other cellular proteins, which appears to be important in CT activation. This novel regulatory action of FC may allow macrophages to adapt to FC loading in atheromata.

Animals↗
PubMed1995

Effect and cellular site of action of cysteine protease inhibitors on the cholesterol esterification pathway in macrophages and Chinese hamster ovary cells.

Stimulation of intracellular cholesterol esterification, which is catalyzed by the enzyme acyl-CoA:cholesterol O-acyltransferase (ACAT), by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. Since other aspects of intracellular cholesterol metabolism involve proteolytic reactions, we looked for evidence of intracellular proteolysis in the stimulation of the cholesterol esterification pathway. When macrophages and CHO cells were incubated with the cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN), the ability of beta-very-low-density lipoprotein (beta-VLDL) and free cholesterol-rich liposomes to stimulate cholesterol esterification was inhibited by 60-90%. Epoxysuccinylleucylamido-3-methylbutane ethyl ester (EST), a cysteine protease inhibitor structurally different from ALLN, also inhibited beta-VLDL-induced cholesterol esterification in CHO cells. The inhibitory effect of the protease inhibitors could not be explained by decreased net expansion of cellular cholesterol pools, inhibition of lipoprotein cholesteryl ester hydrolysis, or blockage of cholesterol trafficking through the lysosomal pathway. Furthermore, stimulation of cholesterol esterification by 25-hydroxycholesterol and sphingomyelinase was not inhibited by ALLN, indicating that ALLN is not acting as a direct ACAT inhibitor in the cells, and suggesting that the ALLN effect is specific for methods of stimulating cholesterol esterification that expand cellular cholesterol pools. Previous studies have shown that inhibition of protein synthesis (e.g., by cycloheximide) stimulates cholesterol esterification in macrophages and CHO cells, suggesting the presence of a short-lived protein inhibitor of cholesterol esterification. Herein, we show that, when added after cycloheximide, ALLN does not inhibit cycloheximide-induced cholesterol esterification in either cell type. The data in this report are consistent with a novel model in which a proteolytic reaction mediates the stimulation of cholesterol esterification specifically by expanded cellular cholesterol pools. The apparent protease-dependent step is not dependent upon lysosomal trafficking of cholesterol and is proximal to the ACAT enzyme itself; it may function by cleaving an endogenous inhibitor of the interaction of expanded cellular cholesterol pools with ACAT.

Animals↗
PubMed1995

The stimulation of the cholesterol esterification pathway by atherogenic lipoproteins in macrophages.

Cholesteryl-ester-loaded macrophages, or foam cells, are prominent features of atherosclerotic lesions and undoubtedly play important roles in lesion development. Foam cell formation involves the uptake of atherogenic lipoproteins or other cholesterol-rich particles by pathways that are down-regulated incompletely or not at all by cholesterol. In addition, postreceptor events that affect intracellular cholesterol metabolism play a critical role in foam cell formation. Increasing evidence shows that the ability of lipoproteins to stimulate cholesterol esterification is dependent upon a regulated and complex pathway that most likely involves one or more proteins in addition to the cholesterol esterifying enzyme itself. The molecular characterization of these proteins, as well as the study of intracellular cholesterol metabolism in vivo, represent important goals for our further understanding of foam cell biology and atherogenesis.

Arteriosclerosis↗
PubMed1994

Effects of particle size on cell uptake of model triglyceride-rich particles with and without apoprotein E.

The effect of apoprotein E on cellular uptake of "VLDL-size" and "IDL-size" triacylglycerol-phospholipid emulsion particles was studied in J-774 macrophages and fibroblasts. In the absence of apoprotein E (apo E), uptake of the smaller IDL-size particles was up to 2-fold higher by mass and 100-fold higher as calculated by particle number. Apo E enhanced the uptake of both VLDL-size and IDL-size emulsion particles, but the effect was greater on the uptake of larger particles (4-5-fold) as compared to up to a 2-fold increase in the uptake of IDL-size particles. In fibroblasts, particle uptake was less than in macrophages (30-50%), but preferential uptake of smaller particles was similarly observed. Particle internalization was demonstrated by 125I-apo E degradation and resistance to particle release by heparin-suramin. In the absence of apo E, cholesteryl ester of emulsion particles (prepared with trace amounts of [3H]cholesteryl ester) was hydrolyzed to free cholesterol, proving internalization and intracellular metabolism. Double-label experiments using DiI-labeled emulsion particles, in the absence and presence of apo E, showed that emulsion particles are rapidly targeted to perinuclear lysosomes. Thus, at physiological concentrations of triglyceride-rich particles, non-receptor-mediated uptake is a mechanism for the uptake of VLDL-size and IDL-size particles into cells.

Apolipoproteins E↗
PubMed1994

Interferon-gamma down-regulates the lipoprotein(a)/apoprotein(a) receptor activity on macrophage foam cells. Evidence for disruption of ligand-induced receptor recycling by interferon-gamma.

Cholesterol loading of macrophages, such as occurs in atheroma foam cells, has recently been shown to upregulate a novel receptor activity that mediates the internalization degradation of the atherogenic lipoprotein, lipoprotein(a) (Lp(a)), and its protein moiety, apoprotein(a), (apo(a)). Herein, the regulation of this receptor activity by macrophage activation and interferon-gamma (IFN-gamma) was investigated. Compared with control foam cells, 125I-recombinant-apo(a) (r-apo(a)) degradation assayed after 5 h of incubation was 3-6-fold less in foam cells derived from thioglycollate- or concanavalin A-elicited mouse peritoneal macrophages. In vitro treatment of foam cells derived from resident mouse peritoneal macrophages or from human monocyte-derived macrophages with IFN-gamma also led to a substantial decrease in the ability of these cells to degrade 125I-rapo(a); similar results were obtained with 125I-Lp(a). In contrast, IFN-gamma-treated foam cells that were incubated for 10 min with 125I-r-apo(a) and then chased for 2 h in the absence of ligand degraded similar amounts of 125I-r-apo(a) as untreated foam cells. To reconcile these data, we hypothesized that the apo(a) receptor activity undergoes ligand-induced recycling and that IFN-gamma disrupts this recycling. To test this idea, control and IFN-gamma-treated foam cells were incubated for 10 min with unlabeled r-apo(a), and then 125I-r-apo(a) receptor activity was assayed at various times thereafter. Untreated foam cells showed clear evidence of ligand-induced recycling of the apo(a) receptor activity, whereas recycling was markedly diminished in the IFN-gamma-treated foam cells. Thus, by disrupting ligand-induced receptor recycling, IFN-gamma leads to down-regulation of the foam cell Lp(a)/apo(a) receptor activity. Since T cells are known to be present in atherosclerotic lesions, these findings raise the possibility that the degradation by atheroma foam cells of Lp(a) and other possible ligands for the receptor may be reversibly regulated by IFN-gamma.

Animals↗
PubMed1994

The actin cytoskeleton is important for the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages.

Stimulation of intracellular cholesterol esterification, which is catalyzed by the enzyme acyl-coenzyme A: cholesterol O-acyltransferase (ACAT), by atherogenic lipoproteins in macrophages is a key step in the ability of these cells to store lipoprotein-cholesterol and in the eventual development of atheroma foam cells. Herein, we provide evidence that the actin cytoskeleton plays an important role in the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. When the actin cytoskeleton of cultured mouse peritoneal macrophages was disrupted by treatment with cytochalasin D or Clostridial C2 toxin, the ability of beta very low density lipoprotein (beta-VLDL) to stimulate cholesterol esterification was decreased 3-6-fold, even under conditions in which beta-VLDL protein degradation, cholesteryl ester hydrolysis, or net cholesterol delivery to the cells was matched. Esterification of cellular phospholipids and triglycerides was not affected by this treatment. Cytochalasin D treatment of macrophages also inhibited the ability of acetyl-low density lipoprotein, another foam cell-forming lipoprotein, to stimulate cholesterol esterification, but stimulation of cholesterol esterification by 25-hydroxycholesterol was not inhibited by cytochalasin D. Cytochalasin D was found to inhibit neither the exit of beta-VLDL-derived cholesterol from lysosomes nor the ability of beta-VLDL to down-regulate endogenous cholesterol synthesis. From these data we conclude that an intact actin cytoskeleton is necessary for efficient stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. Although the exact function of actin in the cholesterol esterification pathway remains to be determined, our data rule out a general role for actin in intracellular cholesterol trafficking or maintenance of ACAT enzyme activity. Rather, we speculate that actin filaments play a role in specific cellular entry processes of atherogenic lipoproteins and/or in establishing transport or contact between the plasma membrane cholesterol substrate pool and the ACAT enzyme in macrophages.

Actins↗
PubMed1994

Free cholesterol loading of macrophages stimulates phosphatidylcholine biosynthesis and up-regulation of CTP: phosphocholine cytidylyltransferase.

Atheroma macrophages accumulate large amounts of free cholesterol (FC) as well as cholesteryl ester (CE). An important adaptive response to FC loading might be increased cellular phospholipid to accommodate the excess FC. To explore this idea, J774 macrophages were incubated for 48 h without lipid, with acetyl-low density lipoprotein to induce mostly CE loading, or with acetyl-low density lipoprotein plus an acyl-CoA:cholesterol O-acyltransferase inhibitor (58035) to induce marked FC loading. The total phospholipid content approximately doubled in FC-loaded versus control or CE-loaded macrophages, with phosphatidylcholine showing the largest increase (approximately 2.5-fold versus control). Electron micrographs revealed the presence of multiple intracellular membrane whorls in the FC-loaded macrophages but not in the control or CE-loaded macrophages. [3H]Choline incorporation into phosphatidylcholine was also greater in FC-loaded macrophages versus control or CE-loaded macrophages, whereas [3H]phosphatidylcholine degradation was similar in all of the macrophages. In these experiments and in others that used non-lipoprotein cholesterol, there was a very close correlation between cellular FC content and phosphatidylcholine biosynthesis. To determine the mechanism of increased phosphatidylcholine synthesis, FC-loaded and CE-loaded macrophages were pulsed with [3H]choline, then chased and assayed for labeled phosphatidylcholine biosynthetic precursors. The only major differences were a 2-fold greater disappearance of label from [3H]choline phosphate and a 5-fold greater appearance of label in CDP-[3H]choline in the FC-loaded macrophages. These data suggest a stimulation of CTP:phosphocholine cytidylyltransferase (CT), which was confirmed by microsomal CT assays. Further studies revealed that the increase in phosphatidylcholine biosynthesis in FC-loaded macrophages was: (a) reversible under conditions of high density lipoprotein3-mediated cellular cholesterol efflux; (b) not blocked by cycloheximide-induced protein synthesis inhibition; and (c) not associated with increased CT mRNA levels. Thus, FC loading of macrophages leads to an increase in phosphatidylcholine mass which is caused by increased phosphatidylcholine biosynthesis. The mechanism appears to be FC-mediated post-translational activation of CT. This adaptive response may be important for atheroma macrophage survival, and disruption of the response may lead to macrophage necrosis and lesion progression.

Animals↗
PubMed1994

Macrophage foam cell lipoprotein(a)/apoprotein(a) receptor. Cell-surface localization, dependence of induction on new protein synthesis, and ligand specificity.

Understanding the interaction of the atherogenic lipoprotein, lipoprotein(a) [Lp(a)], with macrophages may provide important insight into the physiology and pathophysiology of this lipoprotein. We have recently shown that cholesterol loading of macrophages, such as occurs in atheroma foam cells, leads to marked upregulation of a novel receptor activity for native Lp(a) and its plasminogen-like protein component, apoprotein(a) [apo(a)]. We show here that the Lp(a)/apo(a) receptor activity on cholesterol-loaded macrophages is trypsin sensitive, indicating that a cell-surface protein is involved and that the upregulation by cholesterol loading requires new protein synthesis. Ligand studies revealed that the foam cell receptor activity recognizes Lp(a) containing both small and large isoforms of apo(a) as well as rhesus monkey Lp(a), which contains an inactive kringle-4(37) (K4(37) lysine-binding domain. Elastase degradation products of plasminogen did not compete for 125I-labeled recombinant apo(a) [125I-r-apo(a)] internalization and degradation by foam cells, indicating that the K4(37) sequence, as well as the K5 and "protease" domains of apo(a), are not sufficient for receptor interaction. Consistent with these data, the degradation of 125I-r-apo(a) was completely blocked by an anti-Lp(a) polyclonal antibody that does not cross-react with plasminogen. Furthermore, the multiple sialic residues of apo(a) are also not involved in receptor interaction, since desialylated r-apo(a) interacted with foam cells as well as native r-apo(a). In contrast, reduced and denatured r-apo(a) was degraded by foam cells only slightly better than by control cells [28% increased degradation by foam cells versus 450% for native r-apo(a)], suggesting that the upregulated receptor activity recognizes certain secondary and tertiary structural features of apo(a).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Regulation of the threshold for lipoprotein-induced acyl-CoA:cholesterol O-acyltransferase stimulation in macrophages by cellular sphingomyelin content.

Macrophage acyl-CoA:cholesterol O-acyltransferase (ACAT), a key enzyme in atheroma foam cell formation, is stimulated by lipoproteins only after a "threshold" amount of cholesterol has accumulated in the cell. The present study explores the hypothesis that cellular sphingomyelin, by increasing the capacity of the cell to accommodate excess cholesterol, can influence the threshold of ACAT stimulation by lipoproteins. When the sphingomyelin content of macrophages was increased by either incubation with exogenous sphingomyelin or ceramide (a stimulator of endogenous sphingomyelin synthesis), the ability of acetyl-LDL to stimulate whole-cell ACAT activity was substantially reduced despite similar lipoprotein uptake and total cholesterol accumulation as in control cells. When the sphingomyelin content of macrophages was decreased by sphingomyelinase treatment, the ability of acetyl-LDL to stimulate whole-cell ACAT activity was enhanced despite no change in lipoprotein uptake. Importantly, microsomes isolated from control, sphingomyelin-, or sphingomyelinase-treated macrophages showed no difference in ACAT activity when assayed in vitro in the presence of exogenous cholesterol, suggesting that these treatments affected cholesterol trafficking. Lastly, a corollary of the hypothesis, that cells might adapt to a large increase in free cholesterol by increasing their sphingomyelin content, was supported by showing that the sphingomyelin content of macrophages increased 2.6-fold when the cells were induced to accumulate free cholesterol by incubation with acetyl-LDL plus an ACAT inhibitor. Thus, the sphingomyelin content of macrophages can influence the threshold at which ACAT is stimulated by lipoprotein delivery of cholesterol, and the cholesterol content of macrophages can influence the sphingomyelin content of the cell. These findings are consistent with a model in which cellular sphingomyelin plays an important role in accommodating pools of cellular cholesterol that result from the uptake of atherogenic lipoproteins by macrophages.

Animals↗
PubMed1993

Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix. A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation.

Prominent features of atheromata include smooth muscle cells, cholesteryl ester-loaded macrophage foam cells, extracellular matrix, extracellularly trapped and aggregated lipoproteins, and various enzymes including lipoprotein lipase (LpL) and sphingomyelinase (SMase). The interplay of these factors was investigated in cell culture. Incubation of bovine aortic smooth muscle cells for 18 h at 37 degrees C with low density lipoprotein (LDL) in the presence of LpL and SMase led to massive aggregation of LDL on the surface of the cells as viewed by phase, fluorescence (using 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-LDL), and electron microscopy. This aggregation required both enzymes. Studies with 125I-LDL confirmed these observations: 125I-LDL cell association in the presence of LpL plus SMase was 50-100-fold greater than in the absence of the two enzymes and was 10-fold greater than in the presence of either enzyme alone. A similar effect (68-fold enhancement) was seen with 125I-labeled lipoprotein(a) (Lp(a)), another atherogenic lipoprotein. In all cases, 125I-lipoprotein degradation was relatively low (< 5% of cell-associated material). LpL/SMase-mediated association of 125I-LDL with smooth muscle cells was still observed when enzymatically inactive LpL was used. The effect was markedly diminished when the smooth muscle cells were treated with a combination of chondroitin ABC lyase and heparitinase or when mutant Chinese hamster ovary cells that lack cell-surface proteoglycans were used, indicating a specific role for cellular proteoglycans. When smooth muscle cells with 125I-LDL or 125I-Lp(a) aggregates were rinsed and then coincubated with mouse peritoneal macrophages for a further 24 h, visible aggregates disappeared, and there was marked 125I-lipoprotein degradation. Electron micrographs after 24 h of co-culture showed lipid-laden, foamy macrophages situated on top of smooth muscle cells, suggesting that the macrophages phagocytosed and metabolized the smooth muscle cell-associated LDL aggregates. Last, 125I-LDL association with smooth muscle cell extracellular matrix was also synergistically enhanced by LpL and SMase, to a level that was 19-fold greater than in the absence of the two enzymes. Thus, the interaction of LDL and Lp(a) with four atheroma components, namely, smooth muscle cells, extracellular matrix, LpL, and SMase, represents a physiologically plausible mechanism for massive, focal retention and aggregation of atherogenic lipoproteins in the arterial wall with subsequent macrophage foam cell formation.

Animals↗
PubMed1993

Cholesterol loading of macrophages leads to marked enhancement of native lipoprotein(a) and apoprotein(a) internalization and degradation.

Lipoprotein(a) levels in the plasma are strongly correlated with atherosclerotic coronary artery disease. Although the mechanism of this effect is not known, the interaction of lipoprotein(a) with macrophages may be important. Previous work has shown that macrophages in culture internalize and degrade native lipoprotein(a) poorly. In the present study, the interaction of Lp(a) with mouse peritoneal and human monocyte-derived macrophages that were cholesterol-loaded, such as occur in atheromata, was investigated. 125I-Lp(a) degradation was increased 4-5-fold in macrophages that had been loaded with cholesterol by incubation with acetyl-LDL for 2-4 days. The enhanced degradation of Lp(a) by foam cells was chloroquine-sensitive and dependent upon the presence of calcium in the extracellular medium. Incubation of the macrophages with acetyl-LDL plus an inhibitor of acyl CoA:cholesterol acyltransferase, which increased the free cholesterol content and decreased the cholesteryl ester content of the cells, resulted in an even greater up-regulation of 125I-Lp(a) degradation (8-23-fold over control macrophages). The interaction of Lp(a) with cholesterol-loaded macrophages involved the apoprotein(a) moiety of Lp(a) since 125I-apoprotein(a), but not 125I-Lp(a-), was degraded to a much greater extent by foam cells compared to control macrophages. The uptake and degradation of Lp(a) in foam cells was not mediated by LDL, scavenger, LDL receptor-related protein (LRP), or plasminogen receptors. Thus, cholesterol loading of macrophages markedly enhances the internalization and lysosomal degradation of Lp(a) and apo(a) by a calcium-dependent receptor activity different from known lipoprotein receptors.

Animals↗