Beta-very low density lipoprotein is sequestered in surface-connected tubules in mouse peritoneal macrophages

beta-very low density lipoprotein (VLDL) is a large lipoprotein with multiple apoprotein E (apoE) molecules that bind to the LDL receptors on mouse macrophages. Even though they bind to the same receptor, the endocytic processing of beta-VLDL differs from low density lipoprotein (LDL). LDL is rapidly delivered to perinuclear lysosomes and degraded, but much of the beta-VLDL is retained in peripheral compartments for several minutes. We have investigated the properties of these peripheral compartments. Measurement of the pH was made using FITC- phosphatidylethanolamine incorporated into the beta-VLDL, and we found that the peripheral compartments were near neutral in pH. These peripheral, beta-VLDL containing compartments were poorly accessible to antibodies, but a low molecular weight fluorescence quencher (trypan blue) entered the compartments within a few seconds. Intermediate voltage EM of cells labeled with colloidal-gold-beta-VLDL revealed that the peripheral compartments are tubular, surface-connected invaginations. Kinetic studies with fluorescent beta-VLDL showed that the compartments become fully sealed with a half-time of 6 min, and the beta-VLDL is then delivered rapidly to perinuclear lysosomes. By monitoring fluorescence energy transfer between lipid analogs incorporated into the beta-VLDL, some processing of the lipoprotein in the peripheral tubular compartments is demonstrated. The novel mode of uptake of beta-VLDL may account for the high cholesterol ester accumulation induced by this lipoprotein.     

Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux

In atherosclerotic lesions, macrophages store lipid in cytoplasmic inclusions and lysosomes. Regression studies show that lysosomal lipid is not as easily cleared as cytoplasmic inclusion lipid. Macrophages enriched with mildly oxidized low density lipoprotein (oxLDL) accumulate cholesteryl ester (CE) and free cholesterol (FC) in lysosomes. We examined whether lysosomal stores of cholesterol from oxLDL are cleared from THP-1 and mouse macrophages. As in previous studies, oxLDL-enriched THP-1 macrophages accumulated substantial lysosomal cholesterol. Surprisingly, less than 12% of oxLDL-derived lysosomal CE was cleared to efficient FC acceptors (e.g., cyclodextrins, apolipoprotein/phosphatidylcholine vesicles, and fetal bovine serum). Filipin staining showed that lysosomes of oxLDL-treated THP-1 cells contained FC, and despite removal of most of the cell FC (70--80%) by incubation with cyclodextrins, filipin staining of FC in lysosomes did not diminish. Also, when THP-1 macrophages were incubated with [(3)H]CE oxLDL, 73--76% of the [(3)H]CE was retained in a lysosomal hydrolysis resistant pool. In contrast, greater than 90% of acetylated low density lipoprotein (acLDL) [(3)H]CE was hydrolyzed. Furthermore, [(3)H]FC liberated from oxLDL [(3)H]CE was released at a slower rate to cyclodextrins than was [(3)H]FC from acLDL [(3)H]CE. In contrast, only 27% of oxLDL [(3)H]CE was resistant to hydrolysis in mouse macrophages, and the [(3)H]FC generated from oxLDL and acLDL [(3)H]CE was released to cyclodextrins at similar rates. We conclude that lack of hydrolysis and efflux of oxLDL cholesterol is not exclusively inherent in oxLDL, but also requires specific cell factors present in one cell type but not the other.--Yancey, P. G., and W. G. Jerome. Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux. J. Lipid Res. 2001. 42: 317--327.     

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.     

Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein

The mass efflux of free and esterified cholesterol was studied in skin fibroblasts loaded with cholesterol by incubation with low density lipoproteins (LDL) isolated from normal or hypercholesterolemic cynomolgus monkeys. Cells incubated with hypercholesterolemic LDL accumulated 2-3 times more cholesteryl ester than did cells incubated with the same amount of normal LDL. Cholesteryl oleate was the principal cholesteryl ester species to accumulate in cells incubated with both normal and hypercholesterolemic LDL. Efflux of this accumulated cholesterol was absolutely dependent on the presence of a cholesterol acceptor in the culture medium. Lipoprotein-deficient serum (LPDS) was the most potent promoter of cholesterol efflux tested, with maximum efflux occurring at LPDS concentrations greater than 1.5 mg protein/ml. Upon addition of efflux medium containing LPDS, there was a reduction in both the free and esterified cholesterol concentration of the cells. Greater than 90% of the cholesteryl esters that were lost from the cells appeared in the culture medium as free cholesterol, indicating that hydrolysis of cholesteryl esters preceded efflux. Efflux was not inhibited by chloroquine, however, suggesting a mechanism independent of lysosomes. Loss of cellular free cholesterol was maximum by 6 hr and changed very little thereafter up to 72 hr. Cholesteryl ester loss from cells decreased in a log linear fashion for efflux periods of 6-72 hr, with an average half-life for cholesteryl ester efflux of 30 hr, but with a range of 20-50 hr, depending upon the specific cell line. The rate of efflux of cellular cholesteryl esters was similar for cells loaded with normal or hypercholesterolemic LDL. In cells loaded with cholesteryl esters, cholesterol synthesis was suppressed and cholesterol esterification and fatty acid synthesis were enhanced. During efflux, cholesterol synthesis remained maximally suppressed while cholesterol esterification decreased for the first 24 hr of efflux, then plateaued at a level approximately 5-fold higher than control levels, while fatty acid synthesis was slightly stimulated. There was little difference in the rate of efflux of individual cholesteryl ester species. There was, however, the suggestion that reesterification of cholesterol principally to palmitic acid occurred during efflux. Since the rate of cellular cholesteryl ester efflux was similar regardless of whether the cells had been loaded with cholesterol by incubation with normal LDL or hypercholesterolemic LDL, the greater accumulation of cholesterol in cells incubated with hypercholesterolemic LDL cannot be explained by differences in rates of efflux.-St. Clair, R. W., and M. A. Leight. Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein.     

A moderate reduction in extracellular pH protects macrophages against apoptosis induced by oxidized low density lipoprotein

We investigated the effect of pH on macrophage apoptosis induced by oxidized low density lipoprotein (OxLDL), as human atherosclerotic lesions have regions of low pH. Hydroperoxide-rich and oxysterol-rich LDL caused 38% and 74% apoptosis of J774 macrophages, respectively, at 24 h, as measured by the externalization of phosphatidylserine. Native LDL, however, did not cause apoptosis. Reducing the pH of the culture medium from 7.4 to 7.0 inhibited apoptosis induced by hydroperoxide-rich or oxysterol-rich OxLDL by 61% and 46%, respectively (P < 0.001). These data were confirmed by semiquantitative analysis of cytochrome c release from mitochondria. Decreasing the extracellular pH to 7.0 reduced the uptake of hydroperoxide-rich and oxysterol-rich (125)I-labeled LDL by 82% and 42%, respectively, and reduced cell surface binding of oxysterol-rich LDL by 31%. This may explain the reduced apoptosis. Additionally, low pH did not affect OxLDL-induced apoptosis of human monocytes, which do not possess scavenger receptors for OxLDL, but reduced apoptosis of human monocyte-derived macrophages, which do possess them. Our investigations suggest that the presence of areas of low pH within atherosclerotic lesions may reduce the uptake of OxLDL and reduce macrophage apoptosis, thus affecting lesion progression.     

Lysosomal accumulation of oxidized phosphatidylcholine-apolipoprotein B complex in macrophages: intracellular fate of oxidized low density lipoprotein

Oxidized phosphatidylcholine (OxPC) formed in oxidized low density lipoprotein (OxLDL) is thought to be involved in the development of atherosclerosis. OxPC has been found in foam cells in atherosclerotic lesions and suggested to be the epitope for OxLDL recognition by macrophages. OxPC is present as a complex with apolipoprotein B (apoB) in OxLDL, since some OxPC can bind with proteins. In the current study, the intracellular fate of OxPC-apoB complexes after internalization of OxLDL by macrophages was investigated. Murine macrophage cell line J774.1 was incubated with either OxLDL or acetylated LDL for 24 h, then the cells were further incubated for up to 24 h in new medium without lipoprotein. Modified apoB in the cells was quantitated by sandwich ELISA using monoclonal antibodies against OxPC and apoB. Intracellular OxLDL decreased rapidly for the first 4 h to approx. 20% of that before medium change, with the apparent metabolism of OxPC-apoB complex ceasing. OxPC-apoB complexes that remained in the cells after 24 h chasing increased as the period of OxLDL loading in macrophages prolongs. Acetylated LDL in the cells decreased quickly and disappeared after 4 h of chasing. Subcellular fractionation using sucrose density gradient ultracentrifugation of macrophages, which had already accumulated OxPC-apoB complexes by 24 h of incubation with OxLDL and further 24 h chasing, showed that the complex was co-localized with endosomal and lysosomal markers. Immunohistochemical double staining studies demonstrated that OxPC and apoB co-localize in foam cells in early atherosclerotic lesions obtained from human coronary artery. These results suggest that OxPC-apoB complexes originating from OxLDL accumulate in foam cells in human atherosclerotic lesions as well as in macrophages in vitro.     

Variations in transmembrane Ca2+ gradient and apoptosis of macrophages induced by oxidized low density lipoprotein

While Ca²⁺ has been proposed to be a messenger in OxLDL-induced cell death, few studies have addressed the possibility that it may influence the occurrence of apoptosis and necrosis of macrophages induced by OxLDL in virtue of change of transmembrane Ca²⁺ gradient including that across plasma membrane and intracellular organelle membranes. In this paper, various lipophilic Ca²⁺ fluorescent indicators and specific organelle markers were used to study the relationship between the changes of the transmembrane Ca²⁺ gradients and the OxLDL induced apoptosis of macrophages. Our results showed that following exposure of low dose OxLDL to macrophages, the transmembrane Ca²⁺ gradient across the plasma membrane, as well as the membrane-proximal Ca²⁺ gradient, the transnuclear, and the transmitochondrial membrane Ca²⁺ gradient were all changed significantly. These data suggested that changes in transmembrane Ca²⁺ gradients might be involved in the apoptosis of macrophages induced by OxLDL.     

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

Metabolism of oxidized phosphatidylcholines formed in oxidized low density lipoprotein by lecithin-cholesterol acyltransferase.

The possible involvement of lecithin-cholesterol acyltransferase (LCAT) in the metabolism of oxidized phosphatidylcholine (PC) in plasma was investigated. A variety of oxidized products are formed from PC following oxidation of low density lipoproteins (LDL). A significant increase in LDL oxidation levels in patients with familial LCAT deficiency (FLD) has been previously demonstrated by a sensitive sandwich ELISA for oxidized LDL using the monoclonal antibody DLH3 which recognizes oxidized products of PC. In the present study, we found that LCAT produces various metabolites from oxidized PC and that oxidized PC molecules in LDL particles serve as substrates. When the neutral lipid fraction was separated by TLC after the incubation of oxidized 1-palmitoyl-2-[1-14C]linoleoyl PC with human plasma, a number of radioactive bands were formed in addition to cholesteryl ester. These products were not formed from native 1-palmitoyl-2-[1-14C]linoleoyl PC. Plasma from FLD patients also failed to form the additional products from oxidized PC. The addition of dithio-bis(nitrobenzoate) (DTNB), an LCAT inhibitor, or the inactivation of LCAT activity by treating the plasma at 56 degrees C for 30 min abolished the generation of these products from oxidized PC. The activity was recovered in the high density lipoprotein (HDL) fraction but not in the LDL fraction separated from normal plasma. When 1-palmitoyl-2-[1-14C](9-oxononanoyl) PC and 1-stearoyl-2-[1-14C](5-oxovaleroyl)PC, PC oxidation products that contain short chain aldehydes, were incubated with human plasma, radioactive products in the neutral lipid fraction were observed on TLC. LDL containing oxidized PC was measured by sandwich ELISA using an anti-apolipoprotein B antibody and DLH3. The reconstituted oxidized PC-LDL particles were found to have lost their ability to bind DLH3 upon incubation with HDL, while the reactivity of the reconstituted oxidized PC-LDL remained unchanged in the presence of DTNB. These results suggest that LCAT is capable of metabolizing a variety of oxidized products of PC and preventing the accumulation of oxidized PC in circulating LDL particles.     

Engineered polymeric nanoparticles for receptor-targeted blockage of oxidized low density lipoprotein uptake and atherogenesis in macrophages

Strategies to prevent the uptake of modified low density lipoproteins (LDLs) by immune cells, a major trigger of inflammation and atherogenesis, are challenged by complex interfacial factors governing LDL receptor-mediated uptake. We examine a new approach based on a family of "nanoblockers", which are designed to examine the role of size, charge presentation, and architecture on inhibition of highly oxidized LDL (hoxLDL) uptake in macrophages. The nanoblockers are macromolecules containing mucic acid, lauryl chloride, and poly(ethylene glycol) that self-assemble into 15-20 nm nanoparticles. We report that the micellar configuration of the macromolecules and the combined display of anionic (carboxylate) groups in the hydrophobic region of the nanoblockers caused the most effective inhibition in the uptake of hoxLDL by IC21 macrophages. The nanoblockers primarily targeted SR-A and CD36, the major scavenger receptors and modulated the "atherogenic" phenotype of cells in terms of the degree of cytokine secretion, accumulation of cholesterol, and "foam cell" formation. These studies highlight the promise of synthetically engineered nanoblockers against oxidized LDL uptake.     

Proteins derived from platelet alpha granules modulate the uptake of oxidized low density lipoprotein by macrophages.

Activated platelets secrete from their alpha granules a protein-like factor which stimulates the uptake of oxidized low-density lipoprotein (Ox-LDL) by macrophages. The aim of the present study was to evaluate the effect of three purified proteins obtained from platelet alpha granules: platelet-derived growth factor (PDGF), platelet factor-4 (PF-4), and beta-thromboglobulin (B-TG), on the uptake of Ox-LDL by macrophages. Cellular degradation of Ox-LDL by the J-774 A.1 macrophage-like cell line, that was preincubated for 18 h at 37 degrees C, with increasing concentrations of partially purified PDGF, (designated PDGF-CMS-III) was increased by up to 36% in comparison to control cells preincubated without PDGF. This effect was due to PDGF-mediated increase in the number of macrophage receptors for Ox-LDL. The enhanced uptake of Ox-LDL by PDGF resulted in an increase in cellular cholesterol content. Preincubation of macrophages with two types of recombinant PDGF dimers (10 ng/ml), revealed that PDGF-BB stimulated Ox-LDL cellular degradation by 64%, whereas PDGF-AB demonstrated only 34% stimulation, in comparison to control cells that were not treated with PDGF. The stimulatory effect of PDGF-CMS-III and PDGF-AB were reduced by 20% and 28%, respectively, when incubated in the presence of H-7, a specific protein kinase C inhibitor. When macrophages were preincubated with B-TG, cellular uptake of Ox-LDL was reduced by up to 30% at 100 ng B-TG/ml. This effect, however, was obtained only when B-TG was present in the incubation medium. Cellular degradation of Ox-LDL was not affected by preincubation of the cells with PF-4. Pretreatment of PCM with anti-PDGF or anti-B-TG antibodies abolished the effects of PCM on Ox-LDL degradation by macrophages. PDGF, thus, may represent the protein-like factor present in PCM which stimulates Ox-LDL degradation by macrophages, whereas B-TG may have a role in the recognition of PCM particles by the macrophage scavenger receptor. Modulation of macrophage cholesterol content by proteins secreted from activated platelets may have an important role in foam cell formation and atherosclerosis.     

Application of saturation dye 2D-DIGE proteomics to characterize proteins modulated by oxidized low density lipoprotein treatment of human macrophages

Macrophages are believed to play a crucial role in atherogenesis and atherosclerotic plaque progression, mainly through their role in the accumulation of large amounts of cholesteryl ester and foam cell formation after the uptake into the arterial intima of oxidized LDL (oxLDL) particles known to be proatherogenic. The aim of this study was to use a differential proteomic approach to identify the response of human monocyte-derived macrophages after treatment with oxLDL for 24 h. Mass spectrometry analysis (MALDI-TOF) of 2D-DIGE gels made it possible to identify 9 intracellular and 3 secreted proteins that were up-regulated, 11 intracellular and 1 secreted proteins that were down-regulated, and 2 secreted proteins that were induced. This methodological approach not only confirmed the differential expression levels of proteins known to be regulated by oxLDL in macrophages, such as catalase and pyruvate kinase, but also identified oxLDL modulation of other proteins for the first time, including heat shock proteins (HSP) and Actin cytoskeletal proteins. Semiquantitative Western blot confirmed their role. The HSPs identified included heat shock cognate 71 kDa protein (Hsc70), 75 kDa glucose-regulated protein (GRP75), heat shock 70 kDa protein (Hsp70), and 60 kDa (Hsp60) proteins. These highly conserved intracellular protein chaperones, commonly seen in atherosclerotic plaques, appear to participate in protection against cellular stress. Interestingly, oxLDL also modulated several F-Actin capping proteins involved in Actin polymerization and motility: gelsolin, CapG, and CapZ. In conclusion, we have demonstrated the effects of oxLDL in the modulation of several proteins in human macrophages and established a functional profile of the human macrophage during the atherosclerotic process.     

Cholesterol efflux by acute-phase high density lipoprotein: role of lecithin: cholesterol acyltransferase

HDL plays an initial role in reverse cholesterol transport by mediating cholesterol removal from cells. During infection and inflammation, several changes in HDL composition occur that may affect the function of HDL; therefore, we determined the ability of acute-phase HDL to promote cholesterol removal from cells. Acute-phase HDL was isolated from plasma of Syrian hamsters injected with lipopolysaccharide. Cholesterol removal from J 774 murine macrophages by acute-phase HDL was less efficient than that by control HDL because of both a decrease in cholesterol efflux and an increase in cholesterol influx. LCAT activity of acute-phase HDL was significantly lower than that of control HDL. When LCAT activity of control HDL was inactivated, cholesterol efflux decreased and cholesterol influx increased to the level observed in acute-phase HDL. Inactivation of LCAT had little effect on acute-phase HDL. In GM 3468A human fibroblasts, the ability of acute-phase HDL to remove cholesterol from cells was also lower than that of normal HDL. The impaired cholesterol removal, however, was primarily a result of an increase in cholesterol influx without changes in cholesterol efflux. When control HDL in which LCAT had been inactivated was incubated with fibroblasts, cholesterol influx increased to a level comparable to that of acute-phase HDL, without any change in cholesterol efflux. These results suggest that the ability of acute-phase HDL to mediate cholesterol removal was impaired compared with that of control HDL and the lower LCAT activity in acute-phase HDL may be responsible for this impairment. The decreased ability of acute-phase HDL to remove cholesterol from cells may be one of the mechanisms that account for the well-known relationship between infection/inflammation and atherosclerosis.     

Apoptosis induced by oxidized low density lipoprotein in human monocyte-derived macrophages involves CD36 and activation of caspase-3.

Macrophage death may play a crucial role in the progression of atherosclerotic lesions. Here we present evidence that CD36 is involved in oxidized LDL (OxLDL)-induced apoptosis in human monocyte-derived macrophages. Anti-CD36 mAb SMO and OKM-5 reduced the number of apoptotic cells in OxLDL-treated macrophages by more than 94%, but they did not block ceramide-triggered apoptosis. Thrombospondin inhibited the induction of apoptosis by OxLDL in a dose-dependent manner with an IC50 of 10-30 microM. OxLDL did not induce apoptosis in CD36-negative macrophages, demonstrating the essential role of this scavenger receptor in OxLDL-triggered programmed cell death. Neither anti-CD36 Ig nor thrombospondin triggered programmed cell death suggesting that binding to CD36 alone is not sufficient to initiate apoptosis. However, inhibitors of OxLDL-induced apoptosis did not block the uptake of 3H-labeled OxLDL. In contrast, acetylated LDL and polyinosinic acid, ligands of scavenger receptor A (SRA), inhibited uptake of 3H-labeled OxLDL by 65 and 49%, respectively, but did not block OxLDL-induced apoptosis, indicating that SRA is not involved in this process. OxLDL also stimulated caspase-3 activity in human macrophages. Activation of caspase-3 was blocked by anti-CD36 Ig and the caspase-3 inhibitor Z-DEVD-FMK. These results suggest that binding of OxLDL to CD36 initiates a yet unknown OxLDL-specific signaling event, which leads to the rapid activation of caspase-3 resulting in apoptosis of human macrophages. Our data demonstrate a novel role for CD36 in macrophage biology with likely consequences for the development of atherosclerotic lesions.     

Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants

Human serum paraoxonase (PON1) can protect low density lipoprotein (LDL) from oxidation induced by either copper ion or by the free radical generator azo bis amidinopropane hydrochloride (AAPH). During LDL oxidation in both of these systems, a time-dependent inactivation of PON arylesterase activity was observed. Oxidized LDL (Ox-LDL) produced by lipoprotein incubation with either copper ion or with AAPH, indeed inactivated PON arylesterase activity by up to 47% or 58%, respectively. Three possible mechanisms for PON inactivation during LDL oxidation were considered and investigated: copper ion binding to PON, free radical attack on PON, and/or the effect of lipoprotein-associated peroxides on the enzyme. As both residual copper ion and AAPH are present in the Ox-LDL preparations and could independently inactivate the enzyme, the effect of minimally oxidized (Ox-LDL produced by LDL storage in the air) on PON activity was also examined. Oxidized LDL, as well as oxidized palmitoyl arachidonoyl phosphatidylcholine (PAPC), lysophosphatidylcholine (LPC, which is produced during LDL oxidation by phospholipase A2-like activity), and oxidized cholesteryl arachidonate (Ox-CA), were all potent inactivators of PON arylesterase activity (PON activity was inhibited by 35%-61%). PON treatment with Ox-LDL (but not with native LDL), or with oxidized lipids, inhibited its arylesterase activity and also reduced the ability of the enzyme to protect LDL against oxidation. PON Arylesterase activity however was not inhibited when PON was pretreated with the sulfhydryl blocking agent, p-hydroxymercurybenzoate (PHMB). Similarly, on using recombinant PON in which the enzyme's only free sulfhydryl group at the position of cysteine-284 was mutated, no inactivation of the enzyme arylesterase activity by Ox-LDL could be shown. These results suggest that Ox-LDL inactivation of PON involves the interaction of oxidized lipids in Ox-LDL with the PON's free sulfhydryl group. Antioxidants such as the flavonoids glabridin or quercetin, when present during LDL oxidation in the presence of PON, reduced the amount of lipoprotein-associated lipid peroxides and preserved PON activities, including its ability to hydrolyze Ox-LDL cholesteryl linoleate hydroperoxides. We conclude that PON's ability to protect LDL against oxidation is accompanied by inactivation of the enzyme. PON inactivation results from an interaction between the enzyme free sulfhydryl group and oxidized lipids such as oxidized phospholipids, oxidized cholesteryl ester or lysophosphatidylcholine, which are formed during LDL oxidation. The action of antioxidants and PON on LDL during its oxidation can be of special benefit against atherosclerosis since these agents reduce the accumulation of Ox-LDL by a dual effect: i.e. prevention of its formation, and removal of Ox-LDL associated oxidized lipids which are generated during LDL oxidation.     

Intracellular trafficking of pigeon beta-very low density lipoprotein and low density lipoprotein at low and high concentrations in pigeon macrophages

Foam cell formation occurs in vitro at lipoprotein concentrations above 50 microgram/ml in pigeon macrophages. Hypothetically, intracellular trafficking of lipoproteins at higher concentrations may differ from uptake of lipoproteins associated with low concentrations, revealing a separate atherogenic endocytic pathway. Macrophage intracellular trafficking of pigeon beta-very low density lipoprotein (beta-VLDL) and low density lipoprotein (LDL) at low concentrations (12 microgram/ml) near the saturation of high affinity binding sites and high lipoprotein concentrations (50-150 microgram/ml) used to induce foam cell formation were examined. Pigeon beta-VLDL and LDL, differentially labeled with colloidal gold, were added simultaneously to contrast trafficking of beta-VLDL, which causes in vitro foam cell formation, with LDL, which does not. The binding of lipoproteins to cell surface structures, distribution of lipoproteins in endocytic organelles, and the extent of colabeling in the endocytic organelles were determined by thin-section transmission electron microscopy.At low concentrations, the intracellular trafficking of pigeon LDL and beta-VLDL was identical. At high concentrations, LDL was removed more rapidly from the plasma membrane and reached lysosomes more quickly than beta-VLDL. No separate endocytic route was present at high concentrations of beta-VLDL; rather, an increased residence on the plasma membrane, association with nonmicrovillar portions of the plasma membrane, and slower trafficking in organelles of coated-pit endocytosis reflected a more atherogenic trafficking pattern.