Inhibition of cholesteryl ester formation in macrophages by azole antimycotics.

Cultured macrophages take up and metabolize cholesterol-containing liposomes, resulting in massive accumulation of cholesteryl esters in the cells. Using this system, the effects of azole antimycotics on cholesteryl ester formation were studied. Incubation of mouse peritoneal macrophages with ketoconazole, miconazole, or econazole (0.1-10 microM) resulted in concentration-dependent inhibition of cholesteryl ester synthesis from endocytosed cholesterol. IC50 values (concentration resulting in 50% inhibition) were 1.4 +/- 0.1 microM, 4.1 +/- 0.2 microM, and 3.6 +/- 0.2 microM for ketoconazole, miconazole, and econazole, respectively. Complete inhibition was observed with 10 microM ketoconazole, and miconazole and econazole, each at 10 microM, caused 70 and 75% inhibition, respectively, of cholesteryl ester synthesis. The mechanism underlying the inhibition by ketoconazole was further studied. Ketoconazole did not appreciably block the uptake of liposomes or formation of triacylglycerol up to 10 microM. Interestingly, ketoconazole suppressed only 30% of 25-hydroxycholesterol-induced endogenous cholesterol esterification under conditions where esterification of endocytosed cholesterol was completely inhibited. Cytochemical studies with filipin-cholesterol staining revealed that ketoconazole induced massive accumulation of endocytosed cholesterol in macrophage phagolysosomes. These results indicate that ketoconazole inhibits cholesteryl ester formation in macrophages by blocking the intracellular transport of endocytosed cholesterol from lysosomes to the endoplasmic reticulum.     

Propranolol decreases cholesteryl ester accumulation in mouse peritoneal macrophage in vitro

Current evidence indicates that the non-selective beta-blocker propranolol reduces the severity of experimental atherosclerosis in animal model. We studied the effect of propranolol on cholesterol ester synthesis and storage in mouse peritoneal macrophages in vitro. Propranolol in concentrations found in the plasma of patients treated with it (150 ng/ml) decreased by 20-30% beta-VLDL-stimulated synthesis of cholesterol esters with (14C)-oleate. A similar effect was observed also on this synthesis stimulated by acetyl-LDL. In a similar degree the accumulation of cholesteryl esters in the cells was decreased during the 48-hour incubation with lipoproteins in the presence of propranolol. No effect of the drug was observed on the uptake and degradation of beta-VLDL or acetyl-LDL. These observations suggest that propranolol may inhibit cholesteryl ester accumulation in macrophages at the point subsequent to lipoprotein uptake. This may have an impact on the treatment or prevention of atherosclerosis.     

Atorvastatin inhibits ABCA1 expression and cholesterol efflux in THP-1 macrophages by an LXR-dependent pathway

The effect of atorvastatin on adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and cholesterol efflux remains controversial. In an effort to clarify this issue, ABCA1 expression and apolipoprotein AI (apoAI)-mediated cholesterol efflux after atorvastatin treatment were investigated in THP-1 macrophages. Atorvastatin from 2 microM to 40 microM dose-dependently inhibited ABCA1 expression in human monocyte-derived macrophages and phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 monocytes. ApoAI-mediated cholesterol efflux was reduced in PMA-stimulated THP-1 cells treated with atorvastatin, this effect was abolished with acetylated low-density lipoprotein (LDL) pretreatment. Atorvastatin treatment also dose-dependently reduced liver X receptor alpha (LXRalpha) expression and Rho activation. Rho activation by farnysylpyophosphate (FPP) and lysophosphatidic acid (LPA) did not salvage, but further depressed, the cholesterol efflux and ABCA1 expression in the presence of atorvastatin. Without atorvastatin, Rho activation by mevalonate, FPP, and LPA diminished apoAI-mediated cholesterol efflux, and Rho activation by GTPgammaS also decreased ABCA1 messenger ribonucleic acid (mRNA) by 16%. Furthermore, Rho inhibition by C3 exoenzyme increased ABCA1 mRNA by 48% despite a 17% decrease in apoAI-mediated cholesterol efflux. LXRalpha agonists (T01901317 and 22(R)-hydroxycholesterol) prevented any reductions in cholesterol efflux or ABCA1 expression associated with atorvastatin treatment. Furthermore, Western blot analysis demonstrated the reciprocal inhibition of Rho and LXRalpha. In conclusion, atorvastatin decreases ABCA1 expression in noncholesterol-loaded macrophages in an LXRalpha- but not Rho-dependent pathway; this effect can be compromised after acetylated LDL cholesterol loading.     

维登二醇—A抑制胆甾醇酯及其转移蛋白的结合

AIM: To study the wiedendiol-A (W-A) inhibition mechanism of plasma cholesteryl ester (CE) transfer protein (CETP) on the transfer of CE. METHODS: Using gel filtration method. RESULTS: W-A at 30 mumol.L-1 inhibited association of CE with CETP by 76% and CETP transfer activity by 81%. In addition, W-A enhanced binding of TP2, a monoclonal antibody with a CETP C-terminal epitope which is involved in CE binding, to CETP, suggesting a W-A-induced conformational change at the epitope for increased TP2 binding. When CETP activity was measured by varying high-density lipoproteins (HDL) concentration, the apparent Vmax of CE transfer was inhibited by 74% and 83% in the presence of W-A at 14 and 25 mumol.L-1, respectively, while the apparent K(m) of HDL for CETP did not change. CONCLUSION: W-A action is mediated through interaction between W-A and CETP, but not through those between W-A and lipoproteins.     

Reduction of intracellular cholesterol accumulation in THP-1 macrophages by a combination of rosiglitazone and atorvastatin

Rosiglitazone and atorvastatin combination therapy has beneficial effects on both glycemic control and plasma lipid levels in type 2 diabetic patients. In the present study, we sought to determine whether this combination can also exert direct antiatherosclerotic effects in macrophages. Our results show that 2 microM rosiglitazone, alone or combined with 5 microM atorvastatin, significantly upregulated the expression of the ATP-binding cassette transporter ABCA1 and of the class B scavenger receptor CLA-1 (CD36 and LIMPII analog), both involved in cholesterol efflux from macrophages. On the other hand, the combination with atorvastatin attenuated the inductive response elicited by rosiglitazone alone on CD36 mRNA (34%, P < 0.05) and protein (16%, P < 0.05), while the uptake of oxidized low density lipoprotein (LDL) remained unaffected. When we examined the effects of the drugs on acetyl-LDL-induced cholesterol accumulation, we found that only the combination of atorvastatin with rosiglitazone caused a net depletion in the cholesteryl ester content of macrophages (35%, P < 0.05). Our data suggest that this reduction was not mediated by effects on proteins that regulate cholesterol flux, but it may be related to the inhibition of cholesteryl ester formation elicited by the statin.     

Glibenclamide inhibits cholesterol metabolism in macrophage

Sulfonylureas are generally used in the therapeutic treatment of non-insulin-dependent diabetes mellitus. Little is known, however, whether they also affect the lipid metabolism. Using glibenclamide (GB), a typical sulfonylurea, we have investigated its effects on the lipid metabolism in macrophages, J774 and phorbol ester-treated THP-1 cells. In the whole-cell assay system for cholesteryl ester (CE) accumulation that is induced by addition of chemically modified low-density lipoprotein (LDL), such as Ac-LDL and ox-LDL, GB effectively inhibited the CE accumulation of J774 cells in dose-dependent manners. The inhibition was resulted from increase in free cholesterol but not from change in total cholesterol amount. The results suggest that GB acts on acyl-CoA: cholesterol acyltransferase (ACAT) and inhibits its activity. To confirm the possibility, we then tested GB by another assay system using ACAT that was solubilized from the cells and reconstituted into the liposome composed of phosphatidyl choline- cholesterol. GB inhibition was not so much effective as those by CI-976 and NTE-122, known ACAT inhibitors, but the inhibition was complete in the presence of 100 microM GB. Using cell homogenates of PMA-stimulated THP-1 cells, GB also inhibited the ACAT activity to the level of undifferentiated THP-1 cells. These results indicate that GB acts as ACAT inhibitor but the chemical structure is quite different from the conventional ACAT inhibitors, suggesting it can be a seed to generate potential ACAT inhibitors which do not exhibit toxicity in adrenal gland.     

Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1 macrophages

INTRODUCTIONOxidized lipid signaling in macrophages is centralto the pathogenesis of atherosclerosis[1]. Exposure ofmacrophages and other vascular cells to oxidized low-density lipoprotein (ox-LDL) leads to complex changesin gene expression that are collectively thought to influ-ence the development of the atherosclerotic lesion[2].Using two-dimensional gel electrophoresis, the overallprotein map in U937 control cells and U937 foam cellswas obtained. Compared with U937 cells, 37 spots…     

Geranylated flavanone tomentodiplacone B inhibits proliferation of human monocytic leukaemia (THP-1) cells

BACKGROUND AND PURPOSE

Paulownia tomentosa is a rich source of geranylated flavanones, some of which we have previously shown to have cytotoxic activity. To identify members of this class of compounds with cytostatic effects, we assessed the effects of the geranylated flavanone tomentodiplacone B (TOM B) on cell cycle progression and cell cycle regulatory pathways of THP-1 human monocytic leukaemia cells.

EXPERIMENTAL APPROACH

Cell viability was measured by dye exclusion and proliferation by WST-1 assays; cell cycle was monitored by flow cytometry. Regulatory proteins were assessed by immunoprecipitation and kinase assays, and Western blotting.

KEY RESULTS

Tomentodiplacone B had no effect during the first 24 h of cell growth at concentrations between 1 and 2.5 µM, but inhibited cell growth in a dose-dependent manner at concentrations of 5 µM or higher. Growth inhibition during the first 24 h of exposure to TOM B was not accompanied by cytotoxicity as cells were accumulated in G1 phase dose-dependently. This G1 phase accumulation was associated with down-regulation of cyclin-dependent kinase 2 activity and also protein levels of cyclins E1 and A2. However, key stress-related molecules (γ-H2AX, p53 and p21) were not induced, suggesting that TOM B acts by directly inhibiting the cyclin-dependent kinase 2 signalling pathway rather than initiating DNA damage or cellular stress.

CONCLUSIONS AND IMPLICATIONS

Our study provides the first evidence that TOM B directly inhibits proliferation of human monocytic leukaemia cells, and thus is a potential anticancer agent, preventing leukaemia cells from progressing from G1 phase into DNA synthesis.     

PMA诱导THP-1源巨噬细胞的MMP-1表达

目的探讨PMA诱导THP-1源分化成巨噬细胞后不同时间（0、12、24、48、72 h）MMP-1的蛋白和mRNA表达。方法采用佛波醇诱导THP-1细胞分化成巨噬细胞。用酶联免疫吸附实验和逆转录-聚合酶链反应测定不同时间MMP-1蛋白和mR-NA的表达。结果在24 h MMP-1蛋白和mRNA的表达增加（0.545 0±0.008 26,0.048 6±0.010 65;0.118 0±0.013 00,0.091 7±0.008 15;0.190 0±0.001 80,0.165 8±0.784 70;0.142 3±0.116 40,0.127 7±0.009 20;0.095 5±0.002 29,0.054 6±0.022 15;P〈0.05,P〈0.01）。结论 THP-1细胞分化成巨噬细胞后,在24 h MMP-1蛋白和mRNA表达最高,24 h可能降解斑块胶原纤维最强,促进AS的病变的发生和发展。     

胆固醇酯转移蛋白抑制剂开发

脂代谢异常是动脉粥样硬化和冠心病的重要危险因素,调脂药物通过调控脂质代谢,降低血脂水平,进而降低心血管疾病的发生。胆固醇酯转移蛋白(CETP)抑制剂可明显升高高密度脂蛋白-胆固醇,本文综述几种处于临床研究阶段的CETP抑制剂研究进展。     

Comparative study of the effect of beta-blockers with different pharmacological properties on cholesteryl ester formation in mouse peritoneal macrophages

The effect of three beta-blockers: non-selective (propranolol), beta 1-selective (metoprolol), and with intrinsic sympathomimetic activity (pindolol), was investigated on 14C-oleic acid incorporation into cholesteryl esters in mouse peritoneal macrophages. Incorporation of 14C-oleic acid into cholesteryl esters was reduced about 10-fold by propranolol at 10(-4) M while incorporation into triacylglycerols was only 30% decreased at the same concentration. Metoprolol and pindolol had no significant effect on 14C-oleic incorporation into cholesteryl esters or triacylglycerols. Finally, propranolol inhibited the acyl-coenzyme A: cholesterol-O-acyltransferase activity, measured in vitro on macrophages homogenates, while the other studied beta-blockers were ineffective. These results suggest that propranolol could antagonize cholesteryl ester accumulation by macrophages, one of the main processes involved in atherogenesis.     

Exogenous Gas6 attenuates silica-induced inflammation on differentiated THP-1 macrophages

Growth arrest specific 6 (Gas6) has been reported to be related to the modulation of innate immunity. To investigate the potential effect of Gas6 on the regulation of inflammations induced by silica, differentiated THP-1 macrophages were exposed to different concentrations of silica for 6 h and 24 h. Additionally, silica-activated macrophages were treated with Gas6 antibody and Gas6 respectively. Expression levels of Gas6 and inflammatory cytokines (TNF-α, IL-1β and IL-6) were measured. Our results showed that both cell viability and Gas6 expression were suppressed by silica in dose-dependent manners. After pretreatment with Gas6 antibody, silica induced a significant decrease in cell viability and a significant increase in inflammatory cytokines at two time points. Moreover, addition of Gas6 significantly suppressed silica induced TNF-α, IL-1β and IL-6 levels in negative dose-dependent manners, not only in mRNA levels but also in protein levels. Our results suggested that exogenous Gas6 might attenuate inflammations induced by silica on macrophages.     

In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages

Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001–0.01%), and BAK (0.001%–0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.     

Inhibition of cholesteryl ester deposition in macrophages by calcium entry blockers: an effect dissociable from calcium entry blockade.

The effects of calcium entry blockers on stimulated cholesteryl [3H]-oleate deposition in cultured macrophages were characterized in order to elucidate mechanisms underlying possible antiatherosclerotic effects. Stimulation of intracellular cholesteryl [3H]-oleate deposition was initiated by incubation of macrophages with beta-very low density lipoproteins (beta-VLDL). Nifedipine (Class I) markedly reduced cholesteryl [3H]-oleate deposition at all concentrations tested. However, Bay K 8644, a dihydropyridine which is known to stimulate calcium entry, also reduced cholesteryl [3H]-oleate deposition with a similar potency to nifedipine. The effects of three Class II calcium entry blockers were evaluated: verapamil, methoxyverapamil, and diltiazem. Verapamil inhibited cholesteryl [3H]-oleate deposition in a concentration-dependent manner. Similarly, methoxyverapamil reduced cholesteryl [3H]-oleate deposition in a concentration-dependent manner although the reduction was not as great as that produced by verapamil. In contrast, diltiazem at any concentration tested did not inhibit cholesteryl [3H]-oleate deposition. Flunarizine (a Class III calcium entry blocker) produced a modest stimulation of cholesteryl [3H]-oleate deposition at the lowest concentration used (10(-7)M) but marked depression at the highest concentration (10(-5)M). The results indicate calcium entry blockers may exert protective effects on the development of atherosclerosis in animal models of diet-induced hyperlipidaemia by inhibiting intracellular cholesteryl ester deposition, but this effect may not be related to their calcium entry-blocking effects.     

甲壳低聚糖对THP-1源性巨噬细胞脂质转运相关基因表达的影响

目的观察甲壳低聚糖(COS)对离体培养的THP-1源性巨噬细胞脂质转运相关基因表达的影响,以初步探讨COS降低血脂的可能分子机制。方法离体培养人THP-1单核细胞,诱导分化成巨噬细胞后,用100μg/mL的COS处理THP-1源性巨噬细胞6,12,24,48 h,运用半定量RT-PCR方法检测其LDL受体、SR-BⅠ、ABCA1和CD36mRNA表达水平的变化。结果COS处理THP-1源性巨噬细胞24和48 h后,LDL受体mRNA的表达与对照组比较分别下调了15.6%(P<0.05)和30.7%(P<0.01);处理6,12,24,48 h后,SR-BⅠmRNA的表达与对照组比较分别上调了14.7%,23.0%,14.6%(P<0.05)和33.9%(P<0.01);处理6和12 h后,ABCA1 mRNA的表达与对照组比较分别上调了12.6%和14.5%(P<0.05);处理6和12 h后,CD36 mRNA的表达与对照组比较分别下调了25.5%和10.2%(P<0.05)。结论通过细胞离体培养实验表明,COS具有的降低血脂作用可能与其能上调巨噬细胞中AB-CA1和SR-BⅠmRNA表达水平以及降低LDL受体和CD36 mRNA水平有关。     

Autoimmune regulator regulates autophagy in THP-1 human monocytes

The autoimmune regulator (AIRE) is a crucial factor for the induction of central tolerance, and mutations in this gene lead to abnormal immune responses. However, the role of AIRE in autophagy in immune cells, especially in monocytes, is obscure. In the present study, we found that overexpression of AIRE in THP-1 human monocytes resulted in increased endogenous light chain 3 (LC3)-II level and elevated LC3 positive vesicles. Moreover, an autophagy inhibitor or knockdown of AIRE by small interference RNA attenuated these effects. In contrast, the expression of p62/SQSTM1 remained unchanged in THP-1 cells after the corresponding treatment. Our findings indicate that AIRE plays a role in the regulation of autophagy in THP-1 human monocytes.     

Stimulation of human macrophages (THP-1) using Toll-like receptor-2 (TLR-2) agonist decorated nanocarriers

The purpose of this study was to prepare and characterize nanocarrier systems, which allow the application of pDNA vaccines and adjuvants to mucosal vaccination. Chitosan from a vegetal source (Agaricus bisporus) and of GMP quality was used to synthesize the derivative 6-O-carboxymethyl-N,N,N-trimethylchitosan (CM-TMC). Toll-like receptor-2 (TLR-2) agonist, Pam₃Cys, was synthesized and coupled to CM-TMC through a polyethylene glycol (PEG) spacer. Successively, Pam₃Cys decorated nanocarriers were prepared by complexation with plasmid DNA (pDNA) expressing green fluorescence protein (GFP), and characterized with respect to their physicochemical properties and protection of the included plasmid against DNase I enzymatic degradation. In vitro studies using phorbol 12-myristyl 13-acetate (PMA) stimulated macrophage-like THP-1 (mTHP-1) cells were focused on cytotoxicity of both polymers and particles, and their potential to stimulate IL-8 release via the TLR-2 pathway. Our results showed that the TLR-2 functionalized pDNA nanocarriers have the ability to complex and to protect pDNA against enzymatic degradation. pDNA nanocarriers were of around 400?nm in size, and displayed a positive zeta potential of 27.9?±?1.6 mV. Chitosan, CM-TMC, and Pam₃Cys-functionalized CM-TMC polymers displayed cytotoxicity on mTHP1 cells in a concentration-dependent manner, which decreased by 50-fold on complexation with pDNA. In addition, decorated pDNA nanocarriers induced IL-8 secretion by mTHP-1 macrophages, which was increased by 10-fold as compared to nondecorated carriers.     

In vitro uptake of a new cholesteryl carborane ester compound by human glioma cell lines

The cellular uptake and retention of a new cholesteryl carborane ester compound, cholesteryl 1,12-dicarba-closo-dodecaboranel-carboxylate (BCH), by two human glioma cell lines, glioblastoma multiforme SF-763 and SF-767, was evaluated. BCH, which is an extremely hydrophobic compound, was formulated into liposomes and incubated with two human glioma tumor cell lines and one human normal neuron cell line. The amount of BCH uptake by the cells was measured by high performance liquid chromatography. The effects of BCH concentration in the culture medium and the incubation time on the cellular uptake of BCH were studied. In addition, BCH uptake by tumor cells was examined in the presence and absence of lipoprotein in the culture medium. It was found that the amount of BCH taken by the glioma cell lines was much more (up to 14 times) than that by the normal neuron cell line. The cellular uptake of BCH was related to the amount of BCH in the medium as well as the incubation time. The cellular uptake of BCH by SF-763 and SF-767 cells after 16 h of incubation was 283.3 +/- 38.9 and 264.0 +/- 36.5 microg boron/g cells, respectively. The majority of BCH taken up in tumor cells was retained after the subsequent incubation. In the presence of lipoprotein, the cellular uptake of BCH by SF-767 tumor cells was about four times as much as that in the absence of lipoprotein. In conclusion, the cellular uptake of BCH by glioma cells was about 14 times higher than by normal neuron cells. The uptake in glioma cells was up to 10 times higher than that required for successful cancer treatment and BCH was well retained in the tumor cells. Lipoprotein seemed to have an important role in the BCH uptake by glioma cells.