To explore the anti-atherosclerotic mechanism of estrogen and especially observe the effect of estradiol on the content of cholesterol in J774a.1 mouse mononuclear/macrophage-derived foam cells which were incubated with oxidized low-density lipoproteins (ox-LDL). J774a.1 mouse mononuclear/macrophages were incubated with ox-LDL or with both ox-LDL and estradiol (1, 0.1 or 0.01 micromol x L(-1)). Oil red O staining was used to observe the formation of foam cells, and cholesterol oxidase fluorometric was used to determine the content of cellular cholesterol content. Western blotting and RTFQ-PCR were used to observe the expressions of scavenger receptor class B type I (SR-B I ) in J774a.1 foam cells. Compared with the control cells, J774a.1 mouse mononuclear/macrophage-derived foam cells showed significantly increased contents of total cholesterol and cholesterol ester (P < 0.001) and decreased SR-B I mRNA expression (P < 0.01). Estradiol treatment significantly lowered the contents of total cholesterol and cholesterol ester (P < 0.05), and increased SR-B I protein and mRNA expression (P < 0.01) in the foam cells in a dose-dependent manner. Estradiol can inhibit the formation of mononuclear/macrophage-derived foam cells by decreasing the contents of total cholesterol and cholesterol ester and up-regulating the expression of SR-B I in the foam cells.
Animals ; Cell Line ; Cholesterol ; metabolism ; Cholesterol Esters ; metabolism ; Estradiol ; pharmacology ; Foam Cells ; cytology ; metabolism ; Lipoproteins, LDL ; metabolism ; Macrophages ; drug effects ; metabolism ; Mice ; Scavenger Receptors, Class B ; metabolism

Human carboxylesterase 1 (HCE1), belonging to a multigene serine hydrolase family, is a major liver carboxylesterase responsible for the hydrolysis and metabolism of various xenobiotics. It also plays an important role in the transportation and metabolism of endogenous cholesterol ester and free fatty acid, and is closely associated with the pathogenesis of hepatocellular carcinoma. This review describes current developments in the molecular structure, the roles in drug, toxins and lipid metabolism, and the early diagnosis for hepatocellular carcinoma of human carboxylesterase 1.
Carboxylic Ester Hydrolases ; genetics ; physiology ; Carcinoma, Hepatocellular ; diagnosis ; Cholesterol Esters ; metabolism ; Fatty Acids, Nonesterified ; metabolism ; Humans ; Liver Neoplasms ; diagnosis ; Xenobiotics ; metabolism

The purpose of the present study was to investigate the effect of pravastatin on cholesteryl esters in foam cells of murine macrophages and the relation with caveolin-1. RAW 264.7 murine macrophages were coincubated with 80 mg/L oxidized low density lipoprotein (ox-LDL) and pravastatin (0~100 mumol/L) respectively for 24 h. When the best control concentration of pravastatin was confirmed, RAW 264.7 murine macrophages were coincubated with 80 mg/L ox-LDL and pravastatin of the best concentration respectively for 0, 6, 12, 24 h. Oil red O dyeing experiment was used to show the lipid droplets in foam cells. High performance liquid chromatography (HPLC) analysis was performed to determine the content of cellular cholesterol. The level of caveolin-1 was determined by Western blot analysis. The result showed that when macrophages were incubated with 80 mg/L ox-LDL, the ratio of cellular cholesteryl ester to total cholesterol (CE/TC) was beyond 50% through HPLC analysis, and a great deal of lipid droplets displayed in cells through Oil red O dyeing experiment, which manifested the formation of the foam cells. Pravastatin could decrease CE in foam cells in a concentration-dependent manner (1~100 mumol/L). At the concentration of 100 mumol/L, pravastatin decreased cellular CE more than 50%. The effects of pravastatin on the decrease of CE in murine macrophages also displayed a time-dependent manner (incubated with 100 mumol/L pravastatin from 6 to 24 h). Moreover, the expression of caveolin-1 was decreased when the macrophages were incubated with ox-LDL (80 mg/L), while treatment with pravastatin increased the level of caveolin-1 and displayed a concentration- and time-dependent manner. These results suggest that pravastatin could inhibit the development of foam cells through the decrease of cellular CE, which may be related to the upregulation of caveolin-1.
Animals ; Anticholesteremic Agents ; pharmacology ; Caveolin 1 ; metabolism ; Cell Line ; Cells, Cultured ; Cholesterol Esters ; metabolism ; Foam Cells ; metabolism ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; Pravastatin ; pharmacology ; Rats

OBJECTIVE: To observe the effects of immune complexes (IC) prepared from human oxidized density lipoprotein (oxLDL) antibodies and human oxLDL on the foam cell forming and the macrophage activation, and to further uncover the possible mechanisms of immune complexes contributing to the atherosclerosis occurrence. METHODS: The immune complexes of human oxLDL and purified human oxLDL antibodies were added to culture U937 cells by protocols: polyethylene glycol-precipitated insoluble IC (PEG-IC) and IC immobilized by absorption to red blood cells (RBC-IC). With oxLDL as controls and heat-aggregated gamma globulin as an inhibitor of Fc gamma receptor, we measured the cholesterol ester, total cholesterol of the cellular extracts, and quantified the secreted MMP-1 of supernatants from U937 cells. RESULTS: A significant increase of MMP-1 release [(0.769 +/- 0.030) ng/ml vs (0.513 +/- 0.034) ng/ml, P < 0.01] and a higher level of cholesterol ester accumulation [(20.271 +/- 1.668) microg/mg protein vs (17. 226 +/- 1.298 ) microg/mg protein, P < 0.05] in U937 cells incubated with RBC-IC were observed, compared with those incubated with RBC-oxLDL. However, the above quantative difference between the cholesterol ester accumulation induced by oxLDL and insoluble PEG-IC was even more striking, and cholesterol ester accumulation was dosage-dependent. Heat-aggregated gamma globulin (10 mg/ml) as an inhibitor of Fc gamma receptors competitively inhibited cholesterol ester accumulation and decreased PEG-IC stimulating MMP-1 secretion to 71%. CONCLUSION Immune complexe of ox-LDL can transform macrophages into foam cells and activted macrophages. The immunological function of oxLDL is involved in the process of atherosclerosis occurrence.
Antibodies ; pharmacology ; Atherosclerosis ; etiology ; metabolism ; Cholesterol Esters ; metabolism ; Foam Cells ; drug effects ; Humans ; Lipoproteins, LDL ; immunology ; pharmacology ; Macrophage Activation ; drug effects ; Matrix Metalloproteinase 1 ; biosynthesis ; U937 Cells

The lipid droplet (LD) is a unique multi-functional organelle that contains a neutral lipid core covered with a phospholipid monolayer membrane. The LDs have been found in almost all organisms from bacteria to humans with similar shape. Several conserved functions of LDs have been revealed by recent studies, including lipid metabolism and trafficking, as well as nucleic acid binding and protection. We summarized these findings and proposed a hypothesis that the LD is a conserved organelle.
Animals ; Bacteria ; metabolism ; ultrastructure ; Biological Evolution ; Cholesterol Esters ; metabolism ; Humans ; Lipid Droplets ; chemistry ; metabolism ; ultrastructure ; Lipid Metabolism ; genetics ; Nucleic Acids ; metabolism ; Peptide Initiation Factors ; chemistry ; metabolism ; Protein Binding ; RNA-Binding Proteins ; chemistry ; metabolism ; Ribosome Subunits ; chemistry ; metabolism ; Triglycerides ; metabolism

We investigated the mechanism of spontaneous cholesterol efflux induced by acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition, and how an alteration of cholesterol metabolism in macrophages impacts on that in HepG2 cells. Oleic acid anilide (OAA), a known ACAT inhibitor reduced lipid storage substantially by promotion of cholesterol catabolism and repression of cholesteryl ester accumulation without further increase of cytotoxicity in acetylated low-density lipoprotein-loaded THP-1 macrophages. Analysis of expressed mRNA and protein revealed that cholesterol 7alpha-hydroxylase (CYP7A1), oxysterol 7alpha- hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27) were highly induced by ACAT inhibition. The presence of a functional cytochrome P450 pathway was confirmed by quantification of the biliary cholesterol mass in cell monolayers and extracelluar medium. Notably, massively secreted biliary cholesterol from macrophages suppressed the expression of CYP7 proteins in a farnesoid X receptor (FXR)-dependent manner in HepG2 cells. The findings reported here provide new insight into mechanisms of spontaneous cholesterol efflux, and suggest that ACAT inhibition may stimulate cholesterol-catabolic (cytochrome P450) pathway in lesion-macrophages, in contrast, suppress it in hepatocyte via FXR induced by biliary cholesterol (BC).
Anilides/*pharmacology ; Bile/metabolism ; Cells, Cultured ; Cholesterol/*metabolism ; Cholesterol Esters/metabolism ; DNA-Binding Proteins/agonists/*metabolism ; Enzyme Inhibitors/pharmacology ; Gene Expression Regulation, Enzymologic/drug effects ; Hepatocytes/*drug effects/metabolism ; Humans ; Lipid Metabolism/drug effects/genetics ; Macrophages/*drug effects/metabolism ; Models, Biological ; Oleic Acids/*pharmacology ; Receptors, Cytoplasmic and Nuclear/agonists/*metabolism ; Sterol O-Acyltransferase/*antagonists & inhibitors/physiology ; Transcription Factors/agonists/*metabolism

OBJECTIVE: To investigate the expression of Kv1.3 and Kir2.1 during human monocyte-derived macrophages differentiation into foam cells and their function in foam cells formation. METHODS: The human macrophage-derived foam cells were obtained by incubating macrophages with ox-LDL (30 mg/L) for 60 h. The expression of Kv1.3 and Kir2.1 channels were examined by immunocytochemistry, RT-PCR and Western blot. Effects of channel blockers (rMargatoxin and BaCl2) on the cellular cholesterol metabolism were studied by measuring the cellular contents of total cholesterol (TC), free cholesterol (FC), and cholesterol ester (CE) in the presence or absence of the channel blockers. RESULTS: After incubating macrophages with 30 mg/L ox-LDL for 60 h, the cellular contents of TC, FC and CE were markedly increased and the ratio of CE/TC was raised from (14.4+/-6.8)% to (57.9+/-3.5)% (P<0.05), which indicated that the cells had differentiated into foam cells. The expression of Kv1.3 and Kir2.1 channels appeared no obvious difference when differentiating into foam cells (P>0.05); After being blocked specifically (rMargatoxin: 0.1, 10 nmol/L; BaC(12): 75, 125 micromol/L), the cellular contents of TC and CE were markedly reduced without exception and the ratios of CE/TC were all less than 50% (P<0.05). CONCLUSION Both Kv1.3 and Kir2.1 channels play a critical role in differentiation of macrophages into foam cells and blockage of corresponding potassium channels would prevent the formation of the foam cells.
Barium Compounds ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chlorides ; pharmacology ; Cholesterol Esters ; metabolism ; Foam Cells ; cytology ; Humans ; Kv1.3 Potassium Channel ; antagonists & inhibitors ; Macrophages ; cytology ; Monocytes ; cytology ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; Scorpion Venoms ; pharmacology

Both hydropathy plot and in vitro translation results predict the topology of SR-BI; the receptor is an integral membrane protein of 509 amino acids, consisting of a short cytoplasmic N-terminus of 9 amino acids followed by a first transmembrane domain of 22 amino acids, the extracellular domain of 408 amino acids, the second transmembrane domain of 22 amino acids, and the cytoplasmic C-terminus of 47 amino acids. The immunoblot of rBBMV in the presence or absence of pAb589 peptide antigen (the C-terminal 22 amino acid residues of SR-BI) confirmed that the bands at apparent molecular weight of 140 and 210 kDa are SR-BI related protein which might be multimeric forms of SR-BI. 125I apo A-I overlay analysis showed that SR-BI can bind to its ligand, apo A-I, only when it is thoroughly matured - glycosylated and dimerized. The antibody which was generated against extracellular domain of SR-BI (pAb230) not only prevented 125I-labeled apo A-I from binding to 140 kDa band but also inhibited the esterified cholesterol uptake of rabbit BBMV with its IC50 value of 40 microgram/ml of IgG. In contrast, the antibody generated against the C-terminal domain of SR-BI (pAb589) did not show any effect either on cholesterol uptake of rabbit BBMV or 125I-labeled apo A-I binding to 140 kDa band. Overall results show that the ligand binding site of SR-BI in rabbit BBMV is located in extracellular domain, and SR-BI is only functional when it is part of dimeric forms which rationalize the previously found cooperative nature of the binding interaction and maybe a fundamental finding towards the so far poorly understood mechanism of SR-BI function.
Amino Acid Sequence ; Animals ; Antigens, CD36/*metabolism ; Apolipoprotein A-I/metabolism ; Binding Sites/physiology ; Blotting, Western ; Caco-2 Cells ; Cholesterol Esters/metabolism ; Humans ; Intestinal Mucosa/metabolism ; Intestine, Small/*metabolism/ultrastructure ; Iodine Radioisotopes ; Membrane Proteins/*metabolism ; Microvilli/metabolism ; Molecular Sequence Data ; Rabbits ; *Receptors, Immunologic ; Receptors, Lipoprotein/*metabolism ; Receptors, Scavenger ; Scavenger Receptors, Class B ; Surface Properties

The effect of thymic stromal lymphopoietin (TSLP) on macrophage-derived foam cell formation and the underlying mechanism were studied. Macrophages isolated from C57BL/6 mice were co-cultured in vitro with different concentrations of TSLP or TSLPR-antibody in the presence of oxidized low density lipoprotein (ox-LDL). The effects of TSLP on macrophage-derived foam cell formation were observed by using oil red O staining and intracellular lipid determination. The expression levels of foam cell scavenger receptors (CD36 and SRA) as well as ABCA1 and TSLPR were detected by using RT-PCR and Western blotting. As compared with the control group, TSLP treatment significantly promoted lipid accumulation in macrophages, significantly increased protein expression of CD36 and TSLPR in a dose-dependent manner, and significantly reduced the expression of ABCA1 protein in a dose-dependent manner. No significant differences were noted between the TSLPR-antibody group and the control group. TSLP may down-regulate the expression of cholesterol efflux receptor ABCA1 and up-regulate scavenger receptor expression via the TSLPR signaling pathway, thereby promoting macrophage-derived foam cell formation.
ATP Binding Cassette Transporter 1 ; genetics ; metabolism ; Animals ; Antibodies ; immunology ; pharmacology ; Blotting, Western ; CD36 Antigens ; genetics ; metabolism ; Cells, Cultured ; Cholesterol ; metabolism ; Cholesterol Esters ; metabolism ; Cytokines ; pharmacology ; Dose-Response Relationship, Drug ; Foam Cells ; cytology ; drug effects ; metabolism ; Gene Expression ; drug effects ; Immunoglobulins ; immunology ; metabolism ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Receptors, Cytokine ; immunology ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class A ; genetics ; metabolism

AIMTo study the membrane stabilization effect and mechanism of cholesteryl hemisuccinate (CHEMS) on dipalmitoylphosphatidylcholine (DPPC) liposomes; Saikosaponin-D (SSD) liposomes were prepared by using CHEMS as a membrane stabilizer and its encapsulation efficiency and hemolytic activity were evaluated.

METHODSDifferential scanning calorimetry (DSC) and calcein release were used to study membrane stabilization effect of CHEMS on DPPC membrane, Fourier transform infrared spectroscopy (FT-IR) was used to study the interacting mechanism of CHEMS with DPPC, sedimentation experiment was done to study the interaction of CHEMS with SSD and hemolytic study was used to evaluate the hemolytic activity of SSD-liposomes with CHEMS as membrane stabilizer.

RESULTSDSC analysis showed that CHEMS and cholesterol (CHOL) could all decrease the Tm value slightly and the deltaH value markedly. CHEMS was more effective than CHOL in decreasing the deltaH value of DPPC membrane. It suggested that CHEMS was more effective in increasing DPPC membrane stability. It was also proved by calcein release study carried out both in PBS and 30% plasma. The findings by FT-IR suggested that CHEMS has both hydrogen bond and electrostatic interaction with the polar head of DPPC. CHEMS did not form insoluble complex (INCOM) with SSD by sedimentation experiment. Stable SSD-liposomes were prepared using DPPC and CHEMS and decreased effectively the hemolytic activity of SSD, SSD-liposomes may be given intravenously at a concentration of 15 microg x mL(-1), while free SSD was forbidden to be given intravenously.

CONCLUSIONCHEMS was more effective than CHOL in increasing DPPC membrane stability, and it could be of great use in the preparation of cholesterol-dependent hemolytic saponins-liposomes. The hemolytic activity of SSD-liposomes was greatly reduced, allowing a possible concentration of 15 microg x mL(-1) to be intravenously administered.

1,2-Dipalmitoylphosphatidylcholine ; administration & dosage ; Animals ; Calorimetry, Differential Scanning ; Cell Membrane ; drug effects ; Cholesterol ; pharmacology ; Cholesterol Esters ; pharmacology ; Drug Carriers ; Fluoresceins ; metabolism ; Hemolysis ; drug effects ; Liposomes ; Oleanolic Acid ; administration & dosage ; analogs & derivatives ; pharmacology ; Rabbits ; Saponins ; administration & dosage ; pharmacology ; Spectroscopy, Fourier Transform Infrared