Synthetic low-density lipoprotein, a novel biomimetic lipid supplement for serum-free tissue culture

Lipid supplementation in serum-free tissue culture employs solubilization techniques to permit the addition of lipids, but these systems are potentially cytotoxic and do not present lipid in a natural form. In this research a simplified preparation method for synthetic low-density lipoprotein (sLDL) has been developed that involves microfluidization of a solvent lipid solution in a simple aqueous solution. This produces material with size and zeta potential characteristics similar to those of native LDL. sLDL supplementation in tissue culture media provides cholesterol concentrations higher than those achieved by 10% serum supplementation and existing chemically defined lipid supplements. sLDL stimulates NS0 and U937 cellular proliferation in completely serum-free media, the former in a lipid concentration dependent manner that is also related to both the receptor peptide structure employed and its concentration on the particle. The greatest NS0 cellular proliferation was obtained at the highest cholesterol concentration tested (0.5 mg/mL), which was 10 times higher than the cholesterol concentration achieved by standard 10% serum supplementation. U937 cellular proliferation was influenced by variation of sLDL's fatty acid constituents with a natural mixture producing maximal effect. Cell uptake studies in NS0 with fluorescently labeled sLDL indicated that assimilation is reduced by competition from native LDL. The planktonic nature of NS0 cell growth meant that cell binding and uptake experiments were difficult to conduct because of cellular aggregation. However, sLDL-induced U937 proliferation is ablated by the presence of an anti-LDL receptor antibody. The results indicate that sLDL uptake is via the LDL receptor and that sLDL can function as a lipid supplement for serum-free media capable of supplementation to cholesterol concentrations up to 0.5 mg/mL. Cellular uptake studies also suggest that sLDL will be useful for the targeting and delivery of materials to cells. sLDL therefore represents a new and promising synthetic biomimetic alternative to native LDL with multiple applications.     

Antiproliferative efficacy of lipophilic soy isoflavone phytoestrogens delivered by low density lipoprotein particles into cultured U937 cells

Some fat-soluble bioactive substances incorporated into low density lipoprotein (LDL) may be delivered into cells via LDL receptor pathway influencing cellular functions. In this study, we synthesized a number of fat-soluble isoflavone esters and investigated their incorporation into LDL as well as their delivery into U937 cells. Using an artificial transfer system (Celite dispersion), genistein and daidzein oleates and daidzein dilinoleate were efficiently incorporated into LDL with concentrations ranging between 2.7 to 16.9 isoflavone molecules/LDL particle, while much smaller amounts of unesterified isoflavones and genistein stearates were transferred into LDL. LDL containing 7-oleates or 4',7-dioleates of genistein and daidzein significantly reduced U937 cell proliferation by 36-43%. The strongest inhibitory effect was shown by daidzein 4',7-dilinoleate with 93% reduction of cell proliferation. Neither of the 4'-oleates of genistein and daidzein contained in LDLs exhibited antiproliferative effects although they were incorporated into LDL. In summary, we demonstrated that isoflavones made fat-soluble by esterification can be incorporated into LDL in vitro and delivered into cultured U937 cells via the LDL-receptor pathway, reducing the cell proliferation.     

蛋白激酶C抑制剂对U937细胞清道夫受体功能的影响

为了解细胞内蛋白质磷酸化水平对清道夫受体功能的影响,用蛋白激酶Ｃ抑掉剂形孢菌素（ｓｔａｕｒｏｓｐｏｒｉｎｅ,ＳＴＡ）处理人Ｕ９３７细胞,分别测定对照组和处理组细胞对碘标记的氧化低密度脂蛋白（＾１２５Ｉ）ｏｘ－ＬＤＬ的降解,结合,细胞表面受体复合物的内移以及细胞内脂质蓄积的程度,并利用放射自显影方法观察药物对细胞表面受体表达的影响,结果发现ＳＴＡ可以促进细胞结合（＾１２５Ｉ）ｏｘ－ＬＤＬ增加细胞表面     

Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line

The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph. Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth. The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL). U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL. This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor. The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis. Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow. Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis. However, if the cells are incubated in the presence of no less than 40 μg LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated. The effect of LDL is specific since neither VLDL nor HDL₃ at the same protein concentration activates horseradish peroxidase uptake by the cells. Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects. The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish peroxidase binding and uptake by adsorptive endocytosis. The rate of fluid endocytosis and endosome formation seems to depend on cellular cholesterol content presumably because cholesterol is involved in maintaining the appropriate plasma membrane structure and fluidity.     

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.     

Evidence for the expression of the EGF receptor on human monocytic cells

Several malignancies over-express the epidermal growth factor receptor, ligation of which results in cellular differentiation and multiplication. Mononuclear phagocytes secrete this cytokine and its receptor has been detected on microglial cells. This communication describes the expression (and its regulation) of epidermal growth factor receptor (EGFR) on U937 cells. We have shown that a few are EGFR-positive, with expression being up regulated by interleukin 6 (IL-6). Also, when cultured in the presence of serum with the monoclonal anti-EGFR, ICR62, U937s showed a reduced growth rate. By contrast, ICR9 caused a significant increase in cellular proliferation. Both antibodies induced cycle arrest in late G(1)/S phase. When the cells were cultured in the absence of serum, low antibody concentration (10 microg/ml) showed an early inhibitory effect on cell proliferation. By contrast, at high antibody concentrations (50 micro/ml), ICR62 significantly increased the proliferation of U937 cells. We suggest that these results provide indirect evidence for an autocrine action of EGF on U937 cells.     

Effects of reconstituted HDL on charge-based LDL subfractions as characterized by capillary isotachophoresis

Modified LDL in human plasma including small, dense LDL (sdLDL) and oxidized LDL carries a more negative charge than unmodified LDL and is atherogenic. We examined the effects of apolipoprotein A-I (apoA-I)/POPC discs on charge-based LDL subfractions as determined by capillary isotachophoresis (cITP). Three normal healthy subjects and seven patients with metabolic disorders were included in the study. LDL in human plasma was separated into two major subfractions, fast- and slow-migrating LDL (fLDL and sLDL), by cITP. Normal LDL was characterized by low fLDL, and mildly oxidized LDL in vitro and mildly modified LDL in human plasma were characterized by increased fLDL. Moderately oxidized LDL in vitro and moderately modified LDL in a patient with hypertriglyceridemia and HDL deficiency were characterized by both increased fLDL and a new LDL subfraction with a faster mobility than fLDL [very-fast-migrating LDL as determined by cITP (vfLDL)]. cITP LDL subfractions with faster electrophoretic mobility (fLDL vs. sLDL, vfLDL vs. fLDL) were associated with an increased content of sdLDL. Incubation of a plasma fraction with d>1.019 g/ml (depleted of triglyceride-rich lipoproteins) in the presence of apoA-I/POPC discs at 37 degrees C greatly decreased vfLDL and fLDL but increased sLDL. Incubation of whole plasma from patients with an altered distribution of cITP LDL subfractions in the presence of apoA-I/POPC discs also greatly decreased fLDL but increased sLDL. ApoA-I/POPC discs decreased the cITP fLDL level, the free cholesterol concentration, and platelet-activating factor acetylhydrolase activity in the sdLDL subclasses (d=1.040-1.063 g/ml) and increased the size of LDL. ApoA-I/POPC discs reduced charge-modified LDL in human plasma by remodeling cITP fLDL into sLDL subfractions.     

Long time incubation of monocytic U 937 cells with LDL increases specific paf-acether binding and the cellular acetylhydrolase activity.

Besides the well established role of low density lipoproteins (LDL), the phospholipid PAF-acether (paf) seems to be involved in atherogenesis. The effect of LDL (10 micrograms/ml for 24 h, n = 3) on paf binding characteristics of monocyte/macrophage-like U 937 cells was investigated using the radioligand [3H]paf, unlabeled paf and the paf receptor antagonist WEB 2086. The specific [3H]paf binding significantly increased at 1.4 nM (P less than 0.02) and 2.8 nM (P less than 0.01) added [3H]paf with an increased number of paf binding sites in the Scatchard plot analysis of the data. Specific paf binding was functionally active since paf mediated a cellular [Ca2+]i rise. The protein kinase C (PKC) activator PMA (1 nM, 37 degrees C) expressed specific [3H]paf binding already after a 15-min incubation period, indicating a PKC activation as the decisive step of paf receptor expression. LDL also stimulated the paf degrading cellular acetylhydrolase significantly by increasing both Km (9.4 +/- 1.9 vs. 2.0 +/- 0.5 microM, P less than 0.02) and vmax (0.5 +/- 0.2 vs. 0.2 +/- 0.0 nmol/min per mg cell protein, P less than 0.02). The data demonstrate that LDL increases the number of paf receptors on monocyte/macrophage-like U 937 cells and interferes with the dynamics and/or synthesis of the cellular acetyl hydrolase. These effects could be of importance in the pathogenesis of atherosclerosis.     

Oxidized low-density lipoprotein induces macrophage respiratory burst via its protein moiety: A novel pathway in atherogenesis?

Oxidized low-density lipoproteins (oxLDL) play a crucial role in atherogenesis mainly via their capacity to bind and to activate macrophages. However, the role of the protein LDL moiety in this process is not yet established. In this study, human LDL were exposed to hypochlorous acid (HOCl), a selective protein oxidant, or copper sulfate (CuSO(4)), a major lipid oxidant, and tested for their capacity to activate the NADPH-oxidase of human THP-1- and U937-derived macrophages as measured by lucigenin chemiluminescence (CL). Compared to native LDL which had no effect, HOCl-oxLDL triggered potent CL responses in both U937 and THP-1 cells but only when these were fully differentiated into macrophages by phorbol myristate acetate. In contrast, Cu-oxLDL only triggered a moderate CL response of U937 cells and had little effect on THP-1 cells. While delipidation did not affect HOCl-oxLDL-induced CL response it abolished that induced by Cu-oxLDL. Interestingly, U937 cells showed higher CL responses to both types of oxLDL than THP-1 cells, a finding which could be related to their higher expression of the scavenger receptor CD36. Taken together these results strongly support the role of the protein moiety in oxLDL-induced macrophage activation.     

The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts

Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) which contains the LDL receptor (LDLR) binding site (residues 129-169). To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH(2), Ac-hE18A-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.     

白血病抑制因子受体与白血病细胞U937内促分裂原活化蛋白激酶的关系

探讨人白血病细胞系U937白血病抑制因子 (LIF)受体α亚基和另一亚基gp130细胞内区与促分裂原活化蛋白激酶 (MAPK)的关系 ,旨在研究白血病细胞增殖和分化的机制。用基因重组技术将两基因细胞内区互换以构成两嵌合体受体 (190 130 ,130 190 )并分别在U937表达 ,其与野生受体竞争性结合白血病抑制因子 ,用免疫组化和免疫印迹法分析受体细胞内区形成同源性二聚体(190cyt 190cyt,130cyt 130cyt)后的细胞状况和细胞内MAPK的水平。结果表明 ,转染pE190 130后用LIF作用 6h ,U937细胞MAPK表达量增加 ,MAPK形成的二聚体较明显 ,细胞增殖较快 ;而另一嵌合体受体与α亚基形成 190cyt 190cyt时U937细胞MAPK的表达无变化 ,二聚体不明显。说明LIF受体中gp130亚基的细胞内区参与了MAPK的激活及白血病U937细胞增殖信号的传递。     

Phorbol esters inhibit the binding of low-density lipoproteins (LDL) to U-937 monocytelike cells

The present study demonstrates that U-937 monocytelike human cells possess specific LDL receptors. 125I-LDL binds at 4 degrees C on the cell surface. The bound molecules are releasable by heparin. The reaction requires Ca2+ and the binding sites are sensitive to proteolysis. Unlabeled LDL compete with 125I-LDL, whereas HDL are ineffective. At 37 degrees C, LDL are internalized and degraded by a chloroquine-sensitive pathway. Tumor-promoting phorbol esters inhibit the binding of 125I-LDL to its receptor on U-937 cells. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, inhibition is 50% at 5 X 10(-9) M of TPA. After removal of phorbol esters, treated cells recover their 125I-LDL-binding activity in 60 min. The inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the number of available LDL receptors rather than a decrease in receptor affinity.     

Spontaneous production of a suppressor factor by the human macrophage-like cell line U937. I. Suppression of interleukin 1, interleukin 2, and mitogen-induced blastogenesis in mouse thymocytes

The human macrophage-like cell line U937 spontaneously produced a nondialyzable factor that inhibited interleukin 1 (IL 1), interleukin 2 (IL 2), and phytohemagglutinin (PHA)-induced blastogenesis in mouse thymocytes. The suppression by U937 supernatant factor occurred independently of the concentration of IL 1 or PHA, indicating that it was noncompetitive. The U937 suppressor factor was not cytotoxic for thymocytes, nor did it affect the spontaneous proliferation of T lymphoblastoid cell lines and U937. Physicochemical characterization showed that the U937 suppressor factor was nondialyzable, partially inactivated by heat treatment (56 degrees C), ammonium sulfate (67% saturation) precipitable, sensitive to pH 2.5, and resistant to freeze-thawing. Molecular weight of the factor inhibiting co-mitogenic IL 1 activity was approximately 85,000, as estimated by gel filtration. The U937 cell line may provide a model for the study of mechanisms and mediators of immunosuppression by mononuclear phagocytes.     

High‐density lipoprotein acts as an opsonin to enhance phagocytosis of group A streptococcus by U937 cells

We have previously demonstrated that high‐density lipoprotein (HDL) can specifically bind to streptococcal collagen‐like protein 1 (Scl1) of M41‐type group A Streptococcus (GAS). However, the pathological or physiological significance of Scl1?HDL interaction is unknown. Here, the hypothesis that HDL acts as an opsonin to enhance phagocytosis of HDL‐bound GAS by monocytes given that some scavenger receptors can mediate the endocytosis of HDL was tested by using FITC‐labeled bacteria, human U937 monocytes and HDL for phagocytic assays. HDL (10 µg/mL) was found to significantly enhance internalization of M41‐type (ATCC 12373) GAS by U937 cells after 60 min incubation, compared with an HDL‐free group. The internalized GAS were dead after 60 min incubation with U937 cells regardless of presence and absence of HDL. Although very‐low‐density lipoprotein (VLDL) could specifically bind to ATCC 12373 strain, it did not promote phagocytosis of GAS. Additionally, LDL, HDL and VLDL did not enhance phagocytosis of CMCC 32198 strain because this strain did not bind to these lipoproteins. A physiological concentration of HDL (1000 µg/mL) had a similar effect. Anti‐CD36 antibody completely abolished opsonic phagocytosis whereas anti‐CD4 antibody did not, indicating that CD36 is the major scavenger receptor mediating the uptake of HDL‐opsonized GAS by U937 cells. Furthermore, because rScl1 competitively blocked the interaction of ATCC 12373 strain with HDL recombinant Scl1 (rScl1) derived from M41‐type GAS, it significantly decreased opsonophagocytosis of ATCC 12373 strain but not of CMCC 32198 strain. Therefore, our findings suggest that HDL may be an opsonin that enhances CD36‐dependent opsonophagocytosis of GAS by U937 cells.

     

Mechanism of human monocyte activation via the 40-kDa Fc receptor for IgG

It is shown that a mAb specific for the human 40-kDa FcR (FcRII) leads to activation of human monocytic cells but that extensive cross-linking of the receptor is required. Calcium mobilization can be induced in immature monocytic cells (undifferentiated U937 cells) and peripheral blood monocytes with an intact IgG1 anti-FcRII antibody (CIKM5) but not by F(ab')2 fragments of this antibody. The intact antibody can bind in a tripartite manner by its two F(ab') sites and its Fc-binding site whereas the F(ab')2 fragments of this antibody can only bind in a divalent fashion. A rise in intracellular free calcium ion concentration occurs when F(ab')2 fragments are cross-linked with F(ab')2 anti-mouse Ig indicating that more extensive cross-linking of FcRII is required rather than an obligatory requirement for an Fc-FcRII interaction. Calcium mobilization in response to intact or cross-linked F(ab')2 fragments of CIKM5 is associated with superoxide production only in IFN-gamma-primed peripheral blood monocytes and IFN-gamma differentiated U937 cells indicating that the activation signal produced via FcRII is inadequate to fully stimulate non-"primed" cells. A second mAb reactive with FcRII (2E1) does not cause calcium mobilization in monocytes or U937 cells, and partially blocks the effects of CIKM5. 2E1 also blocks CIKM5 superoxide production in IFN-gamma-primed monocytes and differentiated U937 cells. This may be explained in part by the fact that 2E1 is an IgG2a antibody and can only participate in bipartite binding with FcRII. When 2E1 is cross-linked with F(ab')2 anti-mouse Ig there is a small calcium response. This does not cause superoxide generation in IFN-primed monocytes but does do so in IFN-gamma differentiated U937 cells. FcRII is also expressed on granulocytes and some B cells but the effects of cross-linking the receptor on these cells differ from those seen in monocytes.     

Interaction of tryptic peptides of apolipoprotein B-100 with dimyristoylphosphatidylcholine

Apolipoprotein B-100, the major protein constituent of human plasma low-density lipoproteins (LDL), was carboxyamidomethylated, digested with trypsin and the water-soluble tryptic peptides were coincubated with liposomes of dimyristoylphosphatidylcholine (DMPC). At 24.3 degrees C the peptides induced lipid solubilization as evidenced by optical clearing of the lipid-peptide mixture. Lipid-peptide complexes were isolated by density-gradient ultracentrifugation in KBr and had the following properties: DMPC/peptide ratio of 5.6 (w/w); buoyant density of 1.07-1.09 g/ml; discoidal morphology (51 +/- 4 X 260 +/- 28 A) as determined by electron microscopy; and molecular weight of 1.5 X 10(6) as determined by nondenaturing polyacrylamide gel electrophoresis. Compared to liposomes and sonicated vesicles of DMPC, the lipid-peptide complexes had a more rigid structure as assessed by fluorescence polarization. Whereas intact LDL had 42% alpha-helix and 15% beta-pleated sheet, the lipid-peptide complexes contained 70% alpha-helix and less than 5% beta-pleated sheet. The lipid-peptide complexes did not bind to the fibroblast high-affinity LDL receptor. These results show that specific regions in apolipoprotein B-100 which interact with phospholipid have an amphipathic character and may represent primary sites for lipid-protein interaction in LDL.     

Effect of low-density lipoprotein on the expression of high affinity Fc gamma receptors

A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (Fc gamma RI). Measurement of Fc gamma RI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on Fc gamma RI, and monomeric IgG2a, which binds to the ligand binding site of Fc gamma RI. Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability. While incubation of U937 cells with human interferon-gamma (IFN-gamma) increased Fc gamma RI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-gamma resulted in reduced binding of both mAb 32.2 and IgG2a. A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion. Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly. In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density. High-density lipoprotein (HDL3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol. This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of Fc gamma RI, but not for growth. These studies indicate a role for LDL in regulating the expression of Fc gamma RI on the cell surface.     

Vitamin E ameliorates ox-LDL-induced foam cells formation through modulating the activities of oxidative stress-induced NF-κB pathway

The role of antioxidant supplementation with vitamin E in the prevention of atherosclerosis has been a topic of considerable recent interest. The relevance of vitamin E for macrophage-derived foam cell formation, a hallmark of atherosclerosis, however, has not been unequivocally resolved. Here, we investigated the effect of oxidized LDL (ox-LDL) and vitamin E on lipid accumulation and total cholesterol content in U937 macrophages, reactive oxygen species generation and expression of nuclear factor-κB (NF-κB) signaling pathway. The results showed that the mRNA expression and protein levels of P-selectin were evident in U937 macrophages treated with ox-LDL and vitamin E, which indicating that expression of P-selectin is important in macrophage-derived foam cell formation. Moreover, P-selectin changes in ox-LDL-induced foam cell formation can be mediated by vitamin E through activities of nuclear NF-κB activated by serine phosphorylation of NF-κB inhibitor α, suggesting that activation of NF-κB pathway by macrophages may occur. Taken together, these data suggested that vitamin E can prevent ox-LDL-induced foam cell macrophages formation through modulating the activities of oxidative stress-induced NF-κB pathway.