Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (approximately 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.     

Alpha-tocopherol decreases CD36 expression in human monocyte-derived macrophages

Cholesterol-laden macrophages are the hallmark of atherogenesis. The class B scavenger receptor, CD36, binds oxidized low density lipoprotein (OxLDL), is found in atherosclerotic lesions, and is upregulated by OxLDL. We tested the effects of alpha-tocopherol (AT) enrichment of human monocyte-derived macrophages on CD36 expression and cholesteryl ester accumulation. Monocytes isolated from normal volunteers were cultured into macrophages. Macrophages were enriched overnight with various doses of AT (25, 50, and 100 microM). LDL from normal volunteers was oxidized or acetylated (AcLDL) and incubated with macrophages for 48 h at a concentration of 50 or 100 microg/ml. CD36 expression was assessed by flow cytometry. Quantitative analysis of scavenger receptor class A (SR-A) activity was performed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled LDL. CD36 expression was maximal after 8;-10 days of culture. AT (> or =50 microM) significantly decreased CD36 expression upregulated by OxLDL and AcLDL (P < 0.01). Other antioxidants (beta- or gamma-tocopherol) or protein kinase C inhibitors failed to decrease CD36 expression. Concomitantly, DiI-AcLDL and DiI-OxLDL uptake was significantly decreased after AT treatment (P < 0.001). Cholesteryl ester accumulation was significantly decreased after AT enrichment (AcLDL + AT, 77% inhibition; OxLDL + AT, 42% inhibition). In conclusion, AT decreases both CD36 and SR-A expression and cholesteryl ester accumulation in human macrophages. This provides additional scientific support for the antiatherogenic properties of AT.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

Failure of the intracellular itinerary of beta very low density lipoproteins to augment cholesterol esterification in macrophages from Watanabe heritable hyperlipidemic rabbits

beta very low density lipoproteins (beta-VLDL) interact with mouse peritoneal macrophages via specific receptors leading to pronounced stimulation of cholesterol esterification. The present study has defined an alternative pathway for the processing of beta-VLDL in alveolar macrophages from Watanabe heritable hyperlipidemic (WHHL) rabbits. Macrophages from either New Zealand (NZ) or WHHL rabbits degraded 125I-beta-VLDL to an equivalent extent. Degradation was competed to a similar extent in both cell types by either excess unlabeled beta-VLDL or low density lipoprotein, indicative of a specific receptor involvement. Accumulation of intracellular degradation products of beta-VLDL labeled with the residualizing label, dilactitol-125I-tyramine, was similar in both cell types demonstrating that degradation was not due to secreted proteolytic enzymes. beta-VLDL promoted the incorporation of [3H]oleate into cholesteryl-[3H]oleate and increased the cellular mass of cholesterol in NZ macrophages. In contrast, beta-VLDL did not augment cholesteryl-[3H]oleate deposition in WHHL macrophages. This lack of cholesterol esterification occurred despite equivalent acyl-CoA:cholesterol acyltransferase activity in microsomal fractions of both cell types, and similar augmentations in cholesteryl-[3H]oleate during incubation with phospholipase C-treated LDL. Incubation of WHHL macrophages with beta-VLDL increased cellular cholesterol mass, although the response was attenuated compared to NZ cells. To determine whether these disparities in cholesterol esterification were related to the catabolic fate of beta-VLDL-derived cholesterol esters, [3H]cholesteryl oleate was exchanged into the core of beta-VLDL and incubated with macrophages in medium containing [14C]oleate. NZ macrophages accumulated both [3H]cholesterol and [3H]cholesteryl-[14C]oleate after 5 h, indicating hydrolysis and re-esterification of cholesterol esters. In contrast, WHHL macrophages only accumulated [3H]cholesterol esters, suggesting uptake of cholesterol esters without subsequent hydrolysis. These data demonstrate that WHHL macrophages possess a pathway for the intracellular processing of beta-VLDL that permits internalization of the particle without stimulation of cholesterol esterification.     

Impaired ATP-binding cassette transporter A1-mediated sterol efflux from oxidized LDL-loaded macrophages

We investigated the interaction of oxidized low density lipoprotein (OxLDL) with the ATP-binding cassette A1 (ABCA1) pathway in J774 macrophages. Cellular efflux to apolipoprotein AI (apo-AI) of OxLDL-derived cholesterol was lower than efflux of cholesterol derived from acetylated low density lipoprotein (AcLDL). ABCA1 upregulation by 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP) or 22 (R)-hydroxycholesterol (22-OH) and 9-cis retinoic acid (9cRA) increased the efflux to apo-AI of cellular sterols derived from AcLDL, but not of those from OxLDL. AcLDL, but not OxLDL, induced ABCA1 protein content and activity in J774. However, OxLDL did not influence J774 ABCA1 upregulation by cpt-cAMP or 22-OH/9cRA. We conclude that sterols released to cells by OxLDL are available neither as substrate nor as modulator of ABCA1.     

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.     

Different cellular traffic of LDL-cholesterol and acetylated LDL-cholesterol leads to distinct reverse cholesterol transport pathways

Endocytosis of LDL and modified LDL represents regulated and unregulated cholesterol delivery to macrophages. To elucidate the mechanisms of cellular cholesterol transport and egress under both conditions, various primary macrophages were labeled and loaded with cholesterol or cholesteryl ester from LDL or acetylated low density lipoprotein (AcLDL), and the cellular cholesterol traffic pathways were examined. Confocal microscopy using fluorescently labeled 3,3'-dioctyldecyloxacarbocyanine perchlorate-labeled LDL and 1,1'-dioctyldecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate-labeled AcLDL demonstrated their discrete traffic pathways and accumulation in distinct endosomes. ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I) was much greater for AcLDL-loaded macrophages compared with LDL. Treatment with the liver X receptor ligand 22-OH increased efflux to apoA-I in AcLDL-loaded but not LDL-loaded cells. In contrast, at a level equivalent to AcLDL, LDL-derived cholesterol was preferentially effluxed to HDL, in keeping with increased ABCG1. In vivo studies of reverse cholesterol transport (RCT) from cholesterol-labeled macrophages injected intraperitoneally demonstrated that LDL-derived cholesterol was more efficiently transported to the liver and secreted into bile than AcLDL-derived cholesterol. This indicates a greater efficiency of HDL than lipid-poor apoA-I in interstitial fluid in controlling in vivo RCT. These assays, taken together, emphasize the importance of mediators of diffusional cholesterol efflux in RCT.     

Lipoprotein aggregation protects human monocyte-derived macrophages from OxLDL-induced cytotoxicity

Oxidative modifications render low density lipoprotein cytotoxic and enhance its propensity to aggregate and fuse into particles similar to those found in atherosclerotic lesions. We showed previously that aggregation of oxidized LDL (OxLDL) promotes the transformation of human macrophages into lipid-laden foam cells (Asmis, R., and J. Jelk. 2000. Large variations in human foam cell formation in individuals. A fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids. Atherosclerosis. 148: 243-253). Here, we tested the hypothesis that aggregation of OxLDL enhances its clearance by human macrophages and thus may protect macrophages from OxLDL-induced cytotoxicity. We found that increased aggregation of OxLDL correlated with decreased macrophage injury. Using 3H-labeled and Alexa546-labeled OxLDL, we found that aggregation enhanced OxLDL uptake and increased cholesteryl ester accumulation but did not alter free cholesterol levels in macrophages. Acetylated LDL was a potent competitor of aggregated oxidized LDL (AggOxLDL) uptake, suggesting that scavenger receptor A plays an important role in the clearance of AggOxLDL. Inhibitors of actin polymerization, cytochalasin B, cytochalasin D, and latrunculin A, also prevented AggOxLDL uptake and restored OxLDL-induced cytotoxicity. This suggests that OxLDL-induced macrophage injury does not require OxLDL uptake and may occur on the cell surface. Our data demonstrate that aggregation of cytotoxic OxLDL enhances its clearance by macrophages without damage to the cells, thus allowing macrophages to avoid OxLDL-induced cell injury.     

Effects of LDL enriched with different dietary fatty acids on cholesteryl ester accumulation and turnover in THP-1 macrophages

LDL enriched with either saturated, monounsaturated, n-6 polyunsaturated, or n-3 polyunsaturated fatty acids were used to study the effects of dietary fatty acids on macrophage cholesteryl ester (CE) accumulation, physical state, hydrolysis, and cholesterol efflux. Incubation of THP-1 macrophages with acetylated LDL (AcLDL) from each of the four diet groups resulted in both CE and triglyceride (TG) accumulation, in addition to alterations of cellular CE, TG, and phospholipid fatty acyl compositions reflective of the individual LDLs. Incubation with monounsaturated LDL resulted in significantly higher total and CE accumulation when compared with the other groups. After TG depletion, intracellular anisotropic lipid droplets were visible in all four groups, with 71% of the cells incubated with monounsaturated AcLDL containing anisotropic lipid droplets, compared with 30% of cells incubated with n-3 AcLDL. These physical state differences translated into higher rates of both CE hydrolysis and cholesterol efflux in the n-3 group. These data suggest that monounsaturated fatty acids may enhance atherosclerosis by increasing both cholesterol delivery to macrophage foam cells and the percentage of anisotropic lipid droplets, while n-3 PUFAs decrease atherosclerosis by creating more fluid cellular CE droplets that accelerate the rate of CE hydrolysis and the efflux of cholesterol from the cell.     

Pomegranate juice inhibits oxidized LDL uptake and cholesterol biosynthesis in macrophages

Macrophage cholesterol accumulation and foam cell formation are the hallmarks of early atherogenesis. Pomegranate juice (PJ) was shown to inhibit macrophage foam cell formation and development of atherosclerotic lesions. The aim of this study was to elucidate possible mechanisms by which PJ reduces cholesterol accumulation in macrophages. J774.A1 macrophages were preincubated with PJ followed by analysis of cholesterol influx [evaluated as LDL or as oxidized LDL (Ox-LDL) cellular degradation], cholesterol efflux and cholesterol biosynthesis. Preincubation of macrophages with PJ resulted in a significant reduction (P<.01) in Ox-LDL degradation by 40%. On the contrary, PJ had no effect on macrophage degradation of native LDL or on macrophage cholesterol efflux. Macrophage cholesterol biosynthesis was inhibited by 50% (P<.01) after cell incubation with PJ. This inhibition, however, was not mediated at the 3-hydroxy-3 methylglutaryl coenzyme A reductase level along the biosynthetic pathway. We conclude that PJ-mediated suppression of Ox-LDL degradation and of cholesterol biosynthesis in macrophages can lead to reduced cellular cholesterol accumulation and foam cell formation.     

Actin polymerization in macrophages in response to oxidized LDL and apoptotic cells: role of 12/15-lipoxygenase and phosphoinositide 3-kinase

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO-transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages.     

Scavenger receptor class B type I as a receptor for oxidized low density lipoprotein

Scavenger receptor class B type I (SR-BI) has been established as the primary mediator of the selective transfer of lipids from HDL to mammalian cells. In addition to its role in cholesterol metabolism, SR-BI has been shown to bind apoptotic cells and thus could in theory also function as a scavenger receptor. We now show that SR-BI binds oxidized LDL (OxLDL) with high affinity (K(d) of 4.0 +/- 0.5 microg/ml) and mediates internalization and degradation to an extent comparable to that of other scavenger receptors, when normalized to binding activity. The best competitors for OxLDL binding to SR-BI were oxidized lipoproteins, whereas native or acetylated lipoproteins only competed for a small fraction of OxLDL binding. Both the isolated lipids and the isolated protein from OxLDL bound with high affinity to SR-BI and showed partial reciprocal competition. Monoclonal antibody EO6, an antibody against oxidized phospholipids, and 1-palmitoyl-2-(5-oxovaleroyl) phosphatidylcholine (POVPC) both competed effectively with intact OxLDL and with isolated lipids from OxLDL for SR-BI binding.Together, these results demonstrate a potential function of SR-BI, in addition to its role in selective uptake of lipids, to mediate internalization of OxLDL by macrophages and suggest a central role for oxidized phospholipids in this process.     

Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages

The rate of uptake of oxidized low density lipoprotein (LDL) by mouse peritoneal macrophages is similar to that of acetyl LDL; but only approximately 50% of the internalized oxidized LDL is ultimately degraded, in contrast to the near-complete degradation seen with acetyl LDL. The objectives of this study were to determine if this was due to increased surface binding of oxidized LDL, different uptake pathways for oxidized LDL and acetyl LDL, lysosomal dysfunction caused by oxidized LDL, or resistance of oxidized LDL to hydrolysis by lysosomal proteinases. LDL binding studies at 4 degrees C showed that the increased cell association with oxidized LDL could not be explained by differences in cell-surface binding. Immunofluorescence microscopy confirmed intracellular accumulation of apoB-immunoreactive material in macrophages incubated with oxidized LDL, but not with acetyl LDL. The scavenger receptor ligand polyinosinic acid inhibited both the cell association and degradation of oxidized LDL in macrophages by greater than 75%, suggesting a common uptake pathway for degraded LDL and nondegraded LDL. Studies in THP-1 cells also did not reveal more than one specific uptake pathway for oxidized LDL. LDL derivatized by incubation with oxidized arachidonic acid (under conditions that prevented oxidation of the LDL itself) showed inefficient degradation, similar to oxidized LDL. When macrophages were incubated with oxidized LDL together with acetyl 125I-LDL, the acetyl LDL was degraded normally, excluding lysosomal dysfunction as the explanation for the accumulation of oxidized LDL. Generation of trichloroacetic acid-soluble products from oxidized 125I-LDL by exposure to cathepsins B and D was less than that observed with native 125I-LDL. LDL modified by exposure to reactive products derived from oxidized arachidonic acid was also degraded more slowly than native 125I-LDL by cathepsins. In contrast, acetyl 125I-LDL was degraded more rapidly by cathepsins than native 125I-LDL, and aggregated LDL and malondialdehyde-modified LDL were degraded at the same rate as native 125I-LDL. It is concluded that the intracellular accumulation of oxidized LDL in macrophages can be explained at least in part by the resistance of oxidatively modified apolipoprotein B to cathepsins. This resistance to cathepsins does not appear to be due to aggregation of oxidized LDL, but may be a consequence of modification of apolipoprotein B by lipid peroxidation products.     

Relative importance of the LDL receptor and scavenger receptor class B in the beta-VLDL-induced uptake and accumulation of cholesteryl esters by peritoneal macrophages

We investigated the mechanism of beta-very low density lipoprotein (beta-VLDL)-induced foam cell formation derived from peritoneal macrophages from control mice and low density lipoprotein (LDL) receptor-deficient mice to elucidate the role of the LDL receptor in this process. The LDL receptor appeared to be of major importance for beta-VLDL metabolism. Consequently, the accumulation of cholesteryl esters in LDL receptor(-)(/)- macrophages is 2.5-fold lower than in LDL receptor(+)(/)(+) macrophages. In the absence of the LDL receptor, however, beta-VLDL was still able to induce cholesteryl ester accumulation and subsequently we characterized the properties of this residual beta-VLDL recognition site(s) of LDL receptor(-)(/)- macrophages. Although the LDL receptor-related protein is expressed on LDL receptor(-)(/)- macrophages, the cell association of beta-VLDL is not influenced by the receptor-associated protein, and treatment of the macrophages with heparinase and chondroitinase was also ineffective. In contrast, both oxidized LDL (OxLDL) and anionic liposomes were able to inhibit the cell association of (125)I-labeled beta-VLDL in LDL receptor(-)(/)- macrophages by 65%. These properties suggest a role for scavenger receptor class B (SR-B), and indeed, in the LDL receptor(-)(/)- macrophages the selective uptake of cholesteryl esters from beta-VLDL was 2.2-fold higher than that of apolipoproteins, a process that could be inhibited by OxLDL, high density lipoprotein (HDL), and beta-VLDL.In conclusion, the LDL receptor on peritoneal macrophages is directly involved in the metabolism of beta-VLDL and the subsequent foam cell formation. When the LDL receptor is absent, SR-B appears to mediate the remaining metabolism of cholesteryl esters from beta-VLDL.     

Distinct mechanisms for OxLDL uptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI

Modified forms of LDL, including oxidized low density lipoprotein (OxLDL), contribute to macrophage lipid accumulation in the vessel wall. Despite the pathophysiological importance of uptake pathways for OxLDL, the molecular details of OxLDL endocytosis by macrophages are not well understood. Studies in vitro demonstrate that the class B scavenger receptor CD36 mediates macrophage uptake and degradation of OxLDL. Although the closely related scavenger receptor class B type I (SR-BI) binds OxLDL with high affinity, evidence that SR-BI plays a role in OxLDL metabolism is lacking. In this study, we directly compared OxLDL uptake and degradation by CD36 and SR-BI. Our results indicate that although CD36 and SR-BI internalize OxLDL, SR-BI mediates significantly less OxLDL degradation. Endocytosis of OxLDL by both SR-BI and CD36 is independent of caveolae, microtubules, and actin cytoskeleton. However, OxLDL uptake by CD36, but not SR-BI, is dependent on dynamin. The analysis of chimeric SR-BI/CD36 receptors shows that the CD36 C-terminal cytoplasmic tail is necessary and sufficient for dynamin-dependent OxLDL internalization by class B scavenger receptors. These findings indicate that different mechanisms are involved in OxLDL uptake by SR-BI and CD36, which may segregate these two structurally homologous receptors at the cell surface, leading to differences in intracellular trafficking and degradation.     

A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein.

The internalization of oxidized low density lipoprotein (OxLDL) by macrophages is hypothesized to contribute to foam cell formation and eventually to atherosclerotic lesion formation. OxLDL is a ligand for the acetylated low density lipoprotein (AcLDL) receptor, however, our data show that this receptor accounts for less than half of OxLDL uptake by mouse macrophages, suggesting additional receptors for OxLDL. We have developed a novel expression cloning strategy in order to isolate clones encoding OxLDL receptors. In addition to the AcLDL receptor, we isolated a molecular clone for a structurally unrelated receptor capable of mediating the high affinity uptake of OxLDL following transfection into cells. This receptor has been identified as the mouse Fc gamma RII-B2, a member of a family of receptors known to mediate immune complex uptake through recognition of the Fc region of IgG. The uptake of OxLDL by cells transfected with the Fc gamma RII-B2 clone is not blocked by AcLDL but is blocked by the anti-Fc gamma RII monoclonal antibody, 2.4G2.     

Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol condensation.

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. The process involves nonspecific fragmentation, oxidative breakdown, and modification of the lipids and protein of LDL. The process yields a variety of bioactive products, including aldehyde-containing phospholipids, which can cross-react with primary amines (i.e. peptides or phospholipid head groups) to yield Schiff base products. We also demonstrate that such oxidized phospholipid products may further react through a post-oxidation chemical pathway involving aldol condensation. EO6, an IgM monoclonal autoantibody to oxidized phospholipids, blocks the uptake of oxidized LDL (OxLDL) by macrophages. Because the epitope(s) of EO6 also blocks the uptake of OxLDL, a series of oxidized phospholipids, their peptide complexes, and their aldol condensates have been synthesized and characterized, and their antigenicity has been determined. This study defines structural motifs of oxidized phospholipids responsible for antigenicity for EO6. Certain monomeric phospholipids containing short chain fatty acids were antigenic whether oxidized or not in the sn-2 position. However, oxidized phospholipids containing sn-1 long chain fatty acids were not antigenic unless the sn-2 oxidized fatty acid contained an aldehyde that first reacted with a peptide yielding a Schiff base or the sn-2 oxidized fatty acid underwent an aldol type self-condensation. Our data indicate that the phosphorylcholine head group is essential for antigenicity, but its availability depends on the oxidized phospholipid conformation. We suggest that upon oxidation, similar reactions occur in phospholipids on the surface of LDL, generating ligands for macrophage recognition. Synthetic imine adducts of oxidized phospholipids of this type are capable of blocking the uptake of OxLDL.     

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.     

Uptake and accumulation of oxidized low-density lipoprotein during Mycobacterium tuberculosis infection in guinea pigs

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence.     

Beta-amyloid (Abeta40, Abeta42) binding to modified LDL accelerates macrophage foam cell formation

Apart from its role as a risk factor in arteriosclerosis, plasma cholesterol is increasingly recognized to play a major role in the pathogenesis of Alzheimer's disease (AD). Moreover, alterations of intracellular cholesterol metabolism in neuronal and vascular cells are of considerable importance for the understanding of AD. Cellular cholesterol accumulation enhances the deposition of insoluble beta-amyloid peptides, which is considered a hallmark in the pathogenesis of AD. In order to test the hypothesis, whether exogenous beta-amyloid peptides (Abeta42, Abeta40) might contribute to cellular cholesterol accumulation by opsonization of lipoproteins, we compared the binding and uptake of native LDL, enzymatically modified LDL (E-LDL), copper oxidized LDL (Ox-LDL) and HDL as control, preincubated either in the absence or presence of Abeta42 or Abeta40, by human monocytes or monocyte-derived macrophages. Incubation of monocytes and macrophages with Abeta-lipoprotein-complexes lead to increased cellular free and esterified cholesterol when compared to non-opsonized lipoproteins, except for HDL. Furthermore, the cellular uptake of these complexes regulated Abeta-receptors such as FPRL-1 or LRP/CD91. In summary, our results suggest that Abeta42 and Abeta40 act as potent opsonins for LDL, E-LDL and Ox-LDL and enhance cellular cholesterol accumulation as well as Abeta-deposition in vessel wall macrophages.