Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells.

Oxidation of low density lipoprotein (LDL) by cells of the arterial wall or in the presence of copper ions was shown to result in the peroxidation of its fatty acids as well as its cholesterol moiety. LDL incubation with cholesterol oxidase (CO) resulted in the conversion of up to 85% of the lipoprotein unesterified cholesterol (cholest-5-en-3-ol) to cholestenone (cholest-4-en-3-one) in a dose- and time-dependent pattern. Plasma very low density lipoprotein (VLDL) and high density lipoprotein (HDL) could be similarly modified by CO. In cholesterol oxidase-modified LDL (CO-LDL), unlike copper ion-induced oxidized LDL (Cu-Ox-LDL), there was no fatty acids peroxidation, and lipoprotein size or charge as well as LDL cholesteryl ester, phospholipids, and triglycerides content were not affected. CO-LDL, however, demonstrated enhanced susceptibility to oxidation by copper ions in comparison to native LDL. Upon incubation of CO-LDL with J-774 A.1 macrophage-like cell line, cellular uptake and degradation of the lipoprotein was increased by up to 62% in comparison to native LDL but was 15% lower than that of Cu-Ox-LDL. Similarly, the binding of CO-LDL to macrophages increased by up to 80%, and cellular cholesterol mass was increased 51% more than the mass obtained with native LDL. Several lines of evidence indicate that CO-LDL was taken up via the LDL receptor: 1) Excess amounts of unlabeled LDL, but not acetyl-LDL (Ac-LDL), effectively competed with 125I-CO-LDL for the uptake by cells. 2) The degradation of CO-LDL by various types of macrophages and by fibroblasts could be dissociated from that of Ac-LDL and was always higher than that of native LDL. 3) A monoclonal antibody to the LDL receptor (IgG-C7) and a monoclonal antibody to the LDL receptor binding domains on apoB-100 (B1B6) inhibited macrophage degradation of CO-LDL. The receptor for Cu-Ox-LDL, which is not shared with Ac-LDL, was also partially involved in macrophage uptake of CO-LDL, since Cu-Ox-LDL demonstrated some competition capability with CO-125I-LDL for its cellular degradation. CO-LDL cellular degradation was inhibited by chloroquine, thus implying lysosomal involvement in the cellular processing of the lipoprotein. Incubation of macrophages with LDL in the presence of increasing concentrations of cholestenone resulted in up to 52% enhanced lipoprotein cellular degradation suggesting that the cholestenone in CO-LDL might be involved in the enhanced cellular uptake of the modified lipoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Sequestration of acetylated LDL and cholesterol crystals by human monocyte-derived macrophages

Monocyte-derived macrophages accumulate and process cholesterol in atherosclerotic lesions. Because of the importance of this process, we examined the interaction of cholesterol crystals and acetylated low density lipoprotein (AcLDL) with human monocyte-macrophages in a combined chemical and morphological study. These two forms of cholesterol induced extensive compartmentalization of the macrophage cytoplasm. Unexpectedly, the compartments maintained a physical connection to the extracellular space as demonstrated with ruthenium red staining. The compartments formed through invagination of the top surface of the macrophage plasma membrane. Some cholesterol crystals and AcLDL were sequestered within these surface-connected compartments for up to five days in the case of the crystals and for one day in the case of AcLDL. Pulse-chase studies of fractionated macrophages indicated that [3H]cholesterol redistributed from the surface-connected compartments into lysosomes (where the cholesterol remained unesterified) and into lipid droplets (where the cholesterol was stored as cholesteryl ester). Intracellular uptake and esterification of cholesterol was blocked by cytochalasin D. However, once cholesterol was sequestered in the surface-connected compartments, subsequent esterification of the cholesterol could not be inhibited by cytochalasin D. Apolipoprotein E was localized within the surface- connected compartments by immunogold labeling suggesting a possible function for this protein in the processing of lipid taken up through the sequestration pathway. Removal of microcrystalline cholesterol from the medium resulted in release of most of the accumulated cholesterol microcrystals from the macrophages, as well as disappearance of the surface-connected compartments. Thus, sequestration is a novel endocytic mechanism in which endocytic compartments remain connected to the extracellular space. This differs from phagocytosis where endocytic vacuoles rapidly pinch off from the plasma membrane. Sequestration provides a means for macrophages to remove substances from the extracellular space and later release them.     

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.     

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.     

Effects of oxidatively modified LDL on cholesterol esterification in cultured macrophages

Oxidative modification of low density lipoproteins (LDL) has been shown to cause accelerated degradation of LDL via the scavenger receptor pathway in cultured macrophages, and it has been proposed that this process might lead to cholesterol accumulation in macrophages in the arterial wall in vivo. However, oxidation of LDL is accompanied by a substantial reduction in LDL total cholesterol content and hence the amount of cholesterol delivered by oxidatively modified LDL may be less than that delivered by scavenger receptor ligands such as acetyl LDL which results in massive cholesterol accumulation in cultured macrophages. The present studies were done to determine whether the decrease in total cholesterol content during LDL oxidation was due to oxidation of cholesterol and cholesteryl ester, and to determine whether the resulting oxidized sterols could affect cholesterol esterification in cultured macrophages. It was found that when LDL prelabeled with [3H]cholesteryl linoleate was oxidized, there was a decrease in cholesterol mass but no change in radioactivity. The radioactive substances derived from cholesteryl linoleate appeared more polar than the parent compound when analyzed by reverse-phase liquid chromatography, but were not identical with free cholesterol. Thin-layer chromatography of oxidized LDL lipids confirmed the loss of esterified cholesterol, and revealed multiple new bands, some of which matched reference oxysterols including 7-ketocholesterol, 5,6-epoxycholesterol, and 7-hydroxycholesterol. In addition to oxysterols, oxidized cholesteryl esters were also present. Quantitation by gas chromatography indicated that 7-ketocholesterol was the major oxysterol present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation and uptake of LDL by monocyte-derived macrophages from blood of patients with IHD

The main goal of this study was to test our hypothesis that monocyte-derived macrophages of patients with ischemic heart disease (IHD, MP_IHD) are in vivo prestimulated (primed) or stimulated cells. Their capacity for LDL oxidation and uptake exceeds that of macrophages from healthy donors (MP_N). Monocytes were isolated from blood of 18 healthy donors and 25 IHD patients and LDL preparations were obtained from plasma of 16 healthy donors (LDL_N) and 15 patients with familial hypercholesterolemia (LDL_H). Aerobic incubation of LDL_N or LDL_H with MP_IHD resulted in earlier accumulation (by 1 h) of TBARS in LDL, earlier aggregation of LDL (1 h vs 3 h in the case of MP_N), more pronounced LDL apoB fragmentation as well as increased LDL uptake with the increase in accumulation of total cholesterol (TCh; by 1.8–2.1-fold, p < 0.05–0.01) and the decrease in cell viability compared with MP_N (p < 0.01). MP_IHD and MP_N exhibited more effective oxidation and uptake of LDL_H than LDL_N and in most tests this capacity (to oxidize and uptake LDL) increased under hypoxic conditions. These results demonstrate that macrophages of IHD patients are in vivo stimulated cells and that this stimulation, especially in combination with hypercholesterolemic LDL and local or generalized hypoxia, represent serious predisposition for onset or progression of atherosclerosis in IHD patients. The express test model based on MP_IHD may be used for estimation of monocyte/macrophage stimulation in IHD patients as well as for optimization of drug therapy and screening new antiatherosclerotic and antiischemic drugs.     

Reduced uptake of cholesterol esterase-modified low density lipoprotein by macrophages.

Changes in low density lipoprotein (LDL) lipid composition were shown to alter its interaction with the LDL receptor, thus affecting its cellular uptake. Upon incubation of LDL with 5 units/ml cholesterol esterase (CEase) for 1 h at 37 degrees C, there was a 33% reduction in lipoprotein cholesteryl ester content, paralleled by an increment in its unesterified cholesterol. CEase-LDL, in comparison to native LDL, was smaller in size, possessed fewer free lysine amino groups (by 14%), and demonstrated reduced binding to heparin (by 83%) and reduced immunoreactivity against monoclonal antibodies directed toward epitopes along the LDL apoB-100. Incubation of CEase-LDL with the J-774 macrophage-like cell line resulted in about a 30% reduction in lipoprotein binding and degradation in comparison to native LDL, and this was associated with a 20% reduction in macrophage cholesterol mass. Similarly, CEase-LDL degradation by mouse peritoneal macrophages, human monocyte-derived macrophages, and human skin fibroblasts was reduced by 20-44% in comparison to native LDL. CEase-LDL uptake by macrophages was mediated via the LDL receptor and not the scavenger receptor. CEase activity toward LDL was demonstrated in plasma and in cells of the arterial wall such as macrophages and endothelial cells. Thus, CEase modification of LDL may take place in vivo, and this phenomenon may have a role in atherosclerosis.     

Selective uptake and efflux of cholesteryl linoleate in LDL by macrophages expressing 12/15-lipoxygenase

Oxidation of low density lipoprotein (LDL) is a critical step for atherogenesis, and the role of the 12/15-lipoxygenase (12/15-LOX) as well as LDL receptor-related protein (LRP) expressed in macrophages in this process has been suggested. The oxygenation of cholesteryl linoleate in LDL by mouse macrophage-like J774A.1 cells overexpressing 12/15-LOX was inhibited by an anti-LRP antibody but not by an anti-LDL receptor antibody. When the cells were incubated with LDL double-labeled by [3H]cholesteryl linoleate and [125I]apoB, association with the cells of [3H]cholesteryl linoleate expressed as LDL protein equivalent exceeded that of [125I]apoB, indicating selective uptake of [3H]cholesteryl linoleate from LDL to these cells. An anti-LRP antibody inhibited the selective uptake of [3H]cholesteryl ester by 62% and 81% with the 12/15-LOX-expressing cells and macrophages, respectively. Furthermore, addition of LDL to the culture medium of the [3H]cholesteryl linoleate-labeled 12/15-LOX-expressing cells increased the release of [3H]cholesteryl linoleate to the medium in LDL concentration- and time-dependent manners. The transport of [3H]cholesteryl linoleate from the cells to LDL was also inhibited by an anti-LRP antibody by 75%. These results strongly suggest that LRP contributes to the LDL oxidation by 12/15-LOX in macrophages by selective uptake and efflux of cholesteryl ester in the LDL particle.     

Ceramides reduce CD36 cell surface expression and oxidised LDL uptake by monocytes and macrophages

Transglycosylation activity of endo-beta-N-acetylglucosaminidase HS (Endo HS) was investigated using native human transferrin as a donor of an asparagine-linked oligosaccharide and p-nitrophenyl-beta-d-glucose (PNP-beta-d-Glc) as an acceptor of the oligosaccharide. The amount of the product increased dependent on the concentration of the acceptors. Absorption spectrum, exoglycosidase digestion and matrix assisted laser desorption and ionization-time of flight (MALDI-TOF) mass analysis of the transglycosylation product indicated that the asialobiantennary complex type oligosaccharide of human transferrin was transferred to PNP-beta-d-Glc. Endo HS also transferred the oligosaccharide of human transferrin to PNP-alpha-d-Glc, PNP-alpha-d-Gal, PNP-beta-d-Gal, PNP-beta-d-Man, PNP-beta-d-Xyl, PNP-beta-d-GlcNAc, and PNP-glycerol at a different rate. No apparent difference in the K(m) value for human transferrin as an oligosaccharide donor was observed using different acceptors, PNP-beta-d-Glc and PNP-glycerol. The amount of the transglycosylation product successively increased and became constant and then very slightly decreased during the course of enzyme reaction. Endo HS was also transferred the triantennary complex type oligosaccharide of calf fetuin and the bi-, tri-, and tetrantennary complex type oligosaccharides of human alpha(1)-acid glycoprotein to PNP-beta-d-Glc. Furthermore, Endo HS transferred an asparagine-linked oligosaccharide from a hen egg glycopeptide to PNP-beta-d-Glc. The results demonstrate that Endo HS can transfer a wide variety of asparagine-linked complex type oligosaccharides to various monosaccharides. Endo HS was distinct from other enzymes in the specificity for oligosaccharide donors and acceptors.     

Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF)

Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352-2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors.     

Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages

The aim of this work was to investigate the possible mechanisms for uptake by human monocyte-derived macrophages (HMDM) of low density lipoprotein (LDL) pretreated with human arterial chondroitin-6-SO4-rich proteoglycan (LDL-PG). HMDM were incubated with 125I-labeled tyramine cellobiose-labeled LDL-PG, native LDL, and acetylated LDL (Ac-LDL). The results showed that two to four times more LDL-PG than LDL was bound and internalized by the HMDM. Competition experiments showed that LDL-PG competed with native LDL for the apoB,E (LDL) receptor, but not for the Ac-LDL scavenger receptor. Both the LDL and LDL-PG uptake were reduced after preincubation of the macrophages with unlabeled native LDL, though to a lesser extent with LDL-PG. The specific binding of 125I-labeled LDL and 125I-labeled LDL-PG at 4 degrees C was both saturable and concentration-dependent. The dissociation constant (Kd) for binding was 8.6 x 10(-9) M for LDL and 9.4 x 10(-9) M for LDL-PG, but the maximum binding (Bmax) was 1.5-times higher for LDL-PG. Cholesterol derived from LDL-PG was less effective than native LDL in suppressing HMG-CoA reductase activity. The results indicate that the uptake of LDL-PG is mediated not only by the LDL-receptor, but also by another unspecific pathway, which may not be subjected to regulation. These results provide further support for the hypothesis that LDL modifications induced by arterial PG may contribute to the formation of foam cells.     

Cholesterol absorption inhibitor Ezetimibe blocks uptake of oxidized LDL in human macrophages

Ezetimibe belongs to a group of selective and very effective 2-azetidione cholesterol absorption inhibitors which act on the level of cholesterol entry into enterocytes. Recent data indicated that the drug prevents the formation of a heterocomplex consisting of annexin-2 and caveolin-l and leads to specific inhibition of an NPCILI-dependent cholesterol uptake pathway required for uptake of micellar cholesterol into enterocytes. Earlier studies have shown that caveolin-l and annexin-2 are also expressed in human macro-phages and we show in this study that human macrophages express NPC1L1. Moreover in human macrophages, Ezetimibe(SCH58235) and its analogue, SCH354909, are bound to specific cell surface receptors followed by endocytosis via the classical endocytic pathway. SCH58235 had no effect on uptake and/or processing of acetylated LDL (Ac-LDL). In contrast, the compound inhibited uptake of oxidized LDL (Ox-LDL) by -50% in a dose-dependent manner. SCH58235 blocked the lipid-induced induction of LXR/RXR target genes ABCAI, ABCGI, and apolipoprotein E distinctively more effectively in macrophages loaded with Ox-LDL than in those loaded with Ac-LDL. Based on these findings, we presume that the caveolin-l-, annexin-2-, and NPClLI-dependent cholesterol uptake system that is operating in enterocytes may also contribute to class B scavenger receptor-dependent uptake of Ox-LDL in human monocyte-derived macrophages.     

Oxidized LDL induces phosphorylation of non-muscle myosin IIA heavy chain in macrophages

Oxidized LDL (oxLDL) performs critical roles in atherosclerosis by inducing macrophage foam cell formation and promoting inflammation. There have been reports showing that oxLDL modulates macrophage cytoskeletal functions for oxLDL uptake and trapping, however, the precise mechanism has not been clearly elucidated. Our study examined the effect of oxLDL on non-muscle myosin heavy chain IIA (MHC-IIA) in macrophages. We demonstrated that oxLDL induces phosphorylation of MHC-IIA (Ser1917) in peritoneal macrophages from wild-type mice and THP-1, a human monocytic cell line, but not in macrophages deficient for CD36, a scavenger receptor for oxLDL. Protein kinase C (PKC) inhibitor-treated macrophages did not undergo the oxLDL-induced MHC-IIA phosphorylation. Our immunoprecipitation revealed that oxLDL increased physical association between PKC and MHC-IIA, supporting the role of PKC in this process. We conclude that oxLDL via CD36 induces PKC-mediated MHC-IIA (Ser1917) phosphorylation and this may affect oxLDL-induced functions of macrophages involved in atherosclerosis. [BMB Reports 2015; 48(1): 48-53]     

Chondroitin sulfate-modified LDL induces increased cholesteryl ester synthesis and down-regulation of LDL receptors in smooth muscle cells and macrophages

Hypercholesterolemia induces increased transcytosis and accumulation of plasma lipoproteins in the arterial intima, where they interact with matrix proteins and become modified and reassembled lipoproteins. Chondroitin 6-sulfate-modified LDL (CS-mLDL) induces migration, proliferation, and lipid accumulation in human aortic smooth muscle cells (SMCs). To search for the mechanism(s) responsible for lipid accumulation, cultured SMC and macrophages were exposed to CS-mLDL, minimally modified LDL (mmLDL), and native LDL (as a control). Then the cellular uptake, degradation and expression of the LDL receptor (LDL-R) was determined using radioiodinated ligands, ACAT activity assay, fluorescence microscopy and RT-PCR. The uptake of CS-mLDL was 2-fold higher in SMC and 3-to 4-fold higher in macrophages as compared to LDL and mmLDL; the lysosomal degradation of CS-mLDL was slower in SMCs and considerably diminished in macrophages. Compared with LDL, CS-mLDL induced increased synthesis and accumulation of esterified cholesterol in SMCs (∼2-fold) and macrophages (∼10-fold) within an expanded acidic compartment. CS-mLDL and mmLDL down-regulate the gene expression of the LDL-R in the both cell types. Mechanisms of CS-mLDL-induced lipid accumulation in SMC and macrophages involve increased cellular uptake, and diminished cellular degradation that stimulates cholesterol ester synthesis and accumulation in cytoplasmic inclusions and in the lysosomal compartment in an undegraded form; modified lipoproteins induce down-regulation of LDL-R.     

Zinc deficiency does not enhance LDL uptake by P 388 D1 macrophages in vitro

The aim of the study was to investigate the effect of zinc depletion on the susceptibility of Wistar rat low-density lipoproteins (LDL) to peroxidation and their uptake by macrophages, before and after in vitro oxidation. The rats were fed for 7 wk a Zn-adequate diet (100 ppm) ad libitum (AL), a Zn-deficient diet (0.2 ppm) ad libitum (ZD), or a Zn-adequate diet according to the pair-feeding method (PF). Zinc status was determined and, for each group, blood was pooled, and LDL were isolated and labeled with¹²⁵Iodine. An aliquot of each LDL sample was oxidized using Fe^II 10 μM/ascorbate 250 μM. Oxidized and nonoxidized (native) LDL were incubated with P 388 D1 macrophages, and their rates of uptake and degradation by macrophages were measured. Before oxidation, LDL uptake and degradation were not modified by the diet, suggesting that Zn deficiency did not modify rat LDL in vivo. After oxidation, both LDL uptake and degradation were significantly enhanced in the three groups. Nevertheless, we did not observe a significant effect of Zn deficiency. This observation suggests that, in our experimental conditions, Zn deficiency did not modify LDL catabolism.     

Phospholipase A2-modified LDL is taken up at enhanced rate by macrophages.

Modification of the low density lipoprotein (LDL) core or surface lipids were shown to affect the cellular uptake of the lipoproteins and hence the formation of foam cell macrophages. In the present study phospholipase A2 treatment of LDL was shown to produce negatively charged lipoprotein with increased content of lysolechitine. This modified lipoprotein was taken up and degraded by J-774 A.1 macrophage-like cell line at enhanced rate (up to 97% when 10 units/ml of PLase A2 was used) in comparison to control LDL. This effect of PLase A2 was enzyme dose dependent. Competition experiments revealed that the uptake of PLase A2-LDL by the macrophages was specific and was mediated via the LDL receptor. Since PLase A2 was found to exist in various tissues, thus the production of PLase A2-LDL under certain pathological conditions can potentially contribute to foam cell formation and accelerated atherosclerosis.