Cardio-cerebral vascular disease induced by atherosclerosis is a serious cause of human health. The pathogenesis of AS is very complex,and the oxidized low-density lipoprotein( ox LDL) induced foam cells formation is considered to be the most important cytological change in AS. Based on the definition of " TCM chemical biology",we clarified the chemical composition of Ilex hainanensis,the effective substances of I. hainanensis on the activity of anti-AS were screened. Then we found that saponin BF523 had the good inhibitory effect on foam cell formation. In this research,we studied the BF523 as the research object to clarify the molecular target of the active compound of I. hainanensis by foam cell formation model. The results showed that BF523 significantly inhibited the oxidation of ox LDL-induced macrophage foaming and decreased the lipid content in macrophages. BF523 had inhibited the phagocytosis of ox LDL in macrophages by reducing the mRNA and protein levels of scavenger receptor CD36,thereby inhibiting the occurrence and development of AS. These findings not only clarified the mechanism of the inhibition of foam cell formation by saponin BF523,but also provided a useful exploration for the enrichment of the theory of " TCM chemical biology".
Atherosclerosis ; CD36 Antigens ; metabolism ; Cells, Cultured ; Foam Cells ; cytology ; drug effects ; Humans ; Ilex ; chemistry ; Lipoproteins, LDL ; adverse effects

The role of very low density lipoprotein receptor (LVLDR) in the process of foam cell formation was investigated. After the primary cultured mouse peritoneal macrophages were incubated with VLDL, beta-VLDL or low density lipoprotein (LDL), respectively for 24 h and 48 h, foam cells formation was identified by oil red O staining and cellular contents of triglyceride (TG) and total cholesterol (TC) were determined. The mRNA levels of LDLR, LDLR related protein (ILRP) and VLDLR were detected by semi-quantitative RT-PCR. The results demonstrated that VLDL, beta-VLDL and LDL could increase the contents of TG and TC in macrophages. Cells treated with VLDL or beta-VLDL showed markedly increased expression of VLDLR and decreased expression of LDLR, whereas LRP was up-regulated slightly. For identifying the effect of VLDL receptor on cellular lipid accumulation, ldl-A7-VR cells, which expresses VLDLR and trace amount of LRP without functional LDLR, was used to incubate with lipoproteins for further examination. The results elucidated that the uptake of triglyceride-rich lipoprotein mediated by VLDLR plays an important role in accumulation of lipid and the formation of foam cells.
Animals ; Arteriosclerosis ; metabolism ; pathology ; Cells, Cultured ; Cholesterol, LDL ; metabolism ; pharmacology ; Female ; Foam Cells ; cytology ; metabolism ; Lipoproteins, VLDL ; pharmacology ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Receptors, LDL ; metabolism ; Triglycerides ; metabolism

OBJECTIVETo establish the model of bone mesenchymal stem cell-derived smooth muscle cells (BMSC-SMCs) and investigate the role of BMSC-SMCs in the development and progression of artherosclerosis.

METHODSBMSCs were isolated from the femoral bone of SD rats by adherent tissue culture method, and vascular smooth muscle cells (VSMCs) were obtained from the thoracic aorta. The differentiation of BMSCs into BMSC-SMCs was induced in the conditioned medium. The specific markers of BMSCs and BMSC-SMCs were identified by immunofluorescence (IF) staining. After treatment with 80 mg/L oxidative low-density lipoprotein (ox-LDL) for 72 h, the growth characteristics of BMSC-SMCs and VSMCs were observed. Flow cytometry was applied to analyze the cell cycle of BMSC-SMCs and VSMCs.

RESULTSBMCS-SMCs transformed into foam cells after treatment with ox-LDL, which was more obvious in comparison with VSMCs. The growth curve of BMSC-SMCs and VSMCs presented with an S-shape pattern with the cell doubling time of 20 and 32 h, which was reduced to 15 and 28 h after treatment with 80 mg/L ox-LDL, respectively. Flow cytometry showed that exposure to 80 mg/L ox-LDL significantly increased G(0)/G(1) and decreased S and G(2)/M phase cells in both BMSC-SMCs (P<0.01, n=3) and VSMCs (P<0.05, n=3) in comparison with the control cells.

CONCLUSIONBMSC-SMC might be involved in the formation of fatty core and accelerate the development of atherosclerosis.

Animals ; Atherosclerosis ; etiology ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Foam Cells ; cytology ; Lipoproteins, LDL ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; Muscle, Smooth, Vascular ; cytology ; Rats ; Rats, Sprague-Dawley

In order to determine the precise mechanism of the interactions between different types of cells, which are common phenomena in tissues and organs, the importance of coculture techniques are becoming increasingly important. In the area of cardiology, artificial arteries have been developed, based on the understanding of physiological communication of the arterial smooth muscle cells (SMC), endothelial cells (EC), and the extracellular matrix (ECM). In the study of atherosclerosis, the modification of low-density lipoprotein (LDL), which result in the recruitment and accumulation of white blood cells, especially, monocytes/macrophages, and foam cell formation, are hypothesized. Although there are well known animal models, an in vitro model of atherogenesis with a precisely known atherogenesis mechanism has not yet been developed. In this paper, an arterial wall reconstruction model using rabbit primary cultivated aortic SMCs and ECs, was shown. In addition, human peripheral monocytes were used and the transmigration of monocytes was observed by scanning electron and laser confocal microscopy. Monocyte differentiation into macrophages was shown by immunohistochemistry and comprehensive gene expression analysis. With the modified form of LDL, the macrophages were observed to accumulate lipids with a foamy appearance and differentiate into the foam cells in the ECM between the ECs and SMCs in the area of our coculture model.
Animal ; Aorta/physiology* ; Aorta/cytology* ; Arteriosclerosis/etiology* ; Cell Differentiation/physiology ; Cell Movement ; Coculture ; Endothelium, Vascular/physiology ; Endothelium, Vascular/cytology ; Extracellular Matrix/metabolism ; Foam Cells/ultrastructure ; Foam Cells/cytology ; Macrophages/physiology* ; Macrophages/cytology ; Male ; Microscopy, Confocal ; Microscopy, Electron ; Microscopy, Electron, Scanning ; Monocytes/ultrastructure ; Monocytes/physiology ; Muscle, Smooth, Vascular/physiology ; Muscle, Smooth, Vascular/cytology ; Myosin/metabolism ; Protein Isoforms/metabolism ; Rabbits

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

OBJECTIVETo investigate the effect of diclofenac on the expression of Kv1.3 and Kir2.1 channels in human macrophages and the membrane potential and foaming process of the macrophages.

METHODSThe effect of diclofenac on the expression of Kv1.3 and Kir2.1 channels in cultured human monocyte-derived macrophages was investigated using real-time RT-PCR and Western blotting, and its effect on the membrane potential was analyzed with optical mapping of the membrane potential with voltage-sensitive dyes. The ratio of cholesterol ester (CE) in the macrophages following intake of oxidized low-density lipoprotein (OxLDL) was analyzed by an enzymatic fluorometric method.

RESULTSThe expression of Kv1.3 and Kir2.1 channels in the macrophages were down-regulated by diclofenac (1.5 µmol/L and 15 µmol/L). Compared with those in the control group, Kv1.3 mRNA expression was reduced by over 80% and 90% (P<0.05), and Kir2.1 mRNA by over 20% and 30% (P>0.05), respectively; both their protein expression was reduced by over 10% and 60% with a dose- dependent effect (P<0.05). Diclofenac at the two doses dose-dependently reduced the surface fluorescence intensity of the macrophage, and the membrane potential was decreased by 28% and 54%, respectively (P<0.05). Incubation of the macrophages with 30 mg/L OxLDL for 60 h caused an obvious enlargement of the cell volume and deposition of numerous lipid granules in cytoplasm, resulting also in a CE/TC ratio over 50% (P<0.05). Diclofenac at 1.5 and 15 µmol/L both significantly decreased the CE/TC ratio to (23.624∓3.34)% and (13.601∓2.916)% (P<0.05), respectively, but this effect did not show a dose-response relationship (P>0.05).

CONCLUSIONDiclofenac can significant down-regulate the expression of Kv1.3 and Kir2.1 channels in human macrophages, lower their membrane potential and inhibit the process of foam cell formation.

Cells, Cultured ; Diclofenac ; pharmacology ; Foam Cells ; cytology ; drug effects ; Humans ; Kv1.3 Potassium Channel ; metabolism ; Macrophages ; drug effects ; metabolism ; physiology ; Membrane Potentials ; drug effects ; Potassium Channels, Inwardly Rectifying ; metabolism

OBJECTIVETo investigate the potential role of anthocyanins on modulating cholesterol efflux in mouse peritoneal macrophage-derived foam cells and related molecular mechanisms.

METHODSThe macrophages were isolated from pathogen-free NIH mice and were loaded with 50 microg/ml oxLDL for 24 hours, newly formed foam cells were then treated with anthocyanins (cyanidin-3-glucoside, Cy-3-g; or peonidin-3-glucoside, Pn-3-g) at the concentrations of 1 micromol/L, 10 micromol/L, 100 micromol/L for 0 to 36 hours, respectively. The enzymatic-fluorescent method was used to determine cholesterol content in culture medium. ABCA1 expressions at mRNA and protein level were detected by real-time PCR and confocal microscope.

RESULTSCholesterol efflux of macrophage-derived foam cells increased in a time- and dose-dependent manner post anthocyanins treatment. ABCA1 expressions at mRNA and protein levels were also significantly enhanced after anthocyanins treatment in these cells and these effects could be blocked by co-treatment with DIDS, an inhibitor of the transport activities of ABCA1 and blocker of apoAI-mediated cholesterol efflux.

CONCLUSIONThese data demonstrate that anthocyanins induce cholesterol efflux from mouse peritoneal macrophage-derived foam cells via regulating ABCA1 expression.

ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; metabolism ; Animals ; Anthocyanins ; pharmacology ; Cells, Cultured ; Cholesterol ; metabolism ; Foam Cells ; drug effects ; metabolism ; Macrophages, Peritoneal ; cytology ; Mice

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

OBJECTIVETo observe the effect of rosiglitazone on the content of cholesterol and expressions of Acy-coenzyme A: cholesterol acyltransferase 1 (ACAT-1) and scavenger receptor class B type I (SR-BI) in RAW264.7 macrophage-derived foam cells and explore the anti-atherosclerotic mechanism of rosiglitazone.

METHODSRAW264.7 macrophages were incubated with oxidized low-density lipoproteins (ox-LDL) or with both ox-LDL and rosiglitazone (5, 10, or 20 µmol/L). Oil red O staining was used to observe the formation of foam cells, and cholesterol oxidase was used to determine the content of cellular cholesterol contents. Western blotting was used observe the expressions of ACAT-1 and SR-BI in RAW264.7 foam cells.

RESULTSCompared with the control cells, RAW264.7 macrophage-derived foam cells showed significantly increased contents of total cholesterol and free cholesterol (P<0.01) and ACAT-1 expressions (P<0.05) with mildly increased SR-BI expression (P>0.05). Rosiglitazone treatments significantly lowered the contents of total cholesterol and free cholesterol (P<0.05), decreased the expression of ACAT-1 (P<0.05), and increased SR-BI expression (P<0.05) in the foam cells in a dose-dependent manner.

CONCLUSIONRosiglitazone can decrease the contents of total and free cholesterol, down-regulate ACAT-1 expression and up-regulate SR-BI expression in the foam cells produce the anti-atherosclerotic effect.

Acetyl-CoA C-Acetyltransferase ; metabolism ; Cell Line ; Cholesterol ; metabolism ; Foam Cells ; cytology ; drug effects ; metabolism ; Humans ; Scavenger Receptors, Class B ; metabolism ; Thiazolidinediones ; pharmacology

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