Induction of microRNA hsa-let-7d-5p, and repression of HMGA2, contribute protection against lipid accumulation in macrophage 'foam' cells.

Accumulation of excess cholesterol and cholesteryl ester in macrophage 'foam' cells within the arterial intima characterises early 'fatty streak' atherosclerotic lesions, and is accompanied by epigenetic changes, including altered expression of microRNA sequences which determine of gene and protein expression. This study established that exposure to lipoproteins, including acetylated LDL, induced macrophage expression of microRNA hsa-let-7d-5p, a sequence previously linked with tumour suppression, and repressed expression of one of its target genes, high mobility group AT hook 2 (HMGA2). A let-7d-5p mimic repressed expression of HMGA2 (18%; p < 0.05) while a marked increase (2.9-fold; p < 0.05) in expression of HMGA2 was noted in the presence of let-7d-5p inhibitor. Under these conditions, let-7d-5p mimic significantly (p < 0.05) decreased total (10%), free (8%) and cholesteryl ester (21%) mass, while the inhibitor significantly (p < 0.05) increased total (29%) and free cholesterol (29%) mass, compared with the relevant controls. Let-7d-5p inhibition significantly (p < 0.05) increased endogenous biosynthesis of cholesterol (38%) and cholesteryl ester (39%) pools in macrophage 'foam' cells, without altering the cholesterol efflux pathway, or esterification of exogenous radiolabelled oleate. Let-7d-5p inhibition in sterol-loaded cells increased the level of HMGA2 protein (32%; p < 0.05), while SiRNA knockdown of this protein (29%; p < 0.05) resulted in a (21%, p < 0.05) reduction in free cholesterol mass. Thus, induction of let-7d-5p, and repression of its target HMGA2, in macrophages is a protective response to the challenge of increased cholesterol influx into these cells; dysregulation of this response may contribute to atherosclerosis and other disorders such as cancer.

Targeting mitochondrial 18 kDa translocator protein (TSPO) regulates macrophage cholesterol efflux and lipid phenotype.

The aim of the present study was to establish mitochondrial cholesterol trafficking 18 kDa translocator protein (TSPO) as a potential therapeutic target, capable of increasing macrophage cholesterol efflux to (apo)lipoprotein acceptors. Expression and activity of TSPO in human (THP-1) macrophages were manipulated genetically and by the use of selective TSPO ligands. Cellular responses were analysed by quantitative PCR (Q-PCR), immunoblotting and radiolabelling, including [3H]cholesterol efflux to (apo)lipoprotein A-I (apoA-I), high-density lipoprotein (HDL) and human serum. Induction of macrophage cholesterol deposition by acetylated low-density lipoprotein (AcLDL) increased expression of TSPO mRNA and protein, reflecting findings in human carotid atherosclerosis. Transient overexpression of TSPO enhanced efflux (E%) of [3H]cholesterol to apoA-I, HDL and human serum compared with empty vector (EV) controls, whereas gene knockdown of TSPO achieved the converse. Ligation of TSPO (using PK11195, FGIN-1-27 and flunitrazepam) triggered increases in [3H]cholesterol efflux, an effect that was amplified in TSPO-overexpressing macrophages. Overexpression of TSPO induced the expression of genes [PPARA (peroxisome-proliferator-activated receptor α), NR1H3 (nuclear receptor 1H3/liver X receptor α), ABCA1 (ATP-binding cassette A1), ABCG4 (ATP-binding cassette G4) and APOE (apolipoprotein E)] and proteins (ABCA1 and PPARα) involved in cholesterol efflux, reduced macrophage neutral lipid mass and lipogenesis and limited cholesterol esterification following exposure to AcLDL. Thus, targeting TSPO reduces macrophage lipid content and prevents macrophage foam cell formation, via enhanced cholesterol efflux to (apo)lipoprotein acceptors.

Mitochondrial (dys)function and regulation of macrophage cholesterol efflux.

Cholesterol trafficking from the outer to the cholesterol-poor inner mitochondrial membrane requires energized, polarized and actively respiring mitochondria, mediated by a highly regulated multimeric (140-200 kDa) protein complex comprising StAR (steroidogenic acute regulatory protein), mitochondrial TSPO (translocator protein), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide transporter) and associated regulatory proteins. Mitochondrial cholesterol transport is rate-limiting in the CYP27A1 (sterol 27-hydroxylase)-dependent generation of oxysterol ligands for LXR (liver X receptor) transcription factors that regulate the expression of genes encoding proteins in the cholesterol efflux pathway, such as ABC transporters (ATP-binding cassette transporters) ABCA1 and ABCG1. These transporters transfer cholesterol and/or phospholipids across the plasma membrane to (apo)lipoprotein acceptors, generating nascent HDLs (high-density lipoproteins), which can safely transport excess cholesterol through the bloodstream to the liver for excretion in bile. Utilizing information from steroidogenic tissues, we propose that perturbations in mitochondrial function may reduce the efficiency of the cholesterol efflux pathway, favouring accumulation of cholesteryl ester 'foam cells' and allowing the toxic accumulation of free cholesterol at the interface between the endoplasmic reticulum and the mitochondrial membrane. In turn, this will trigger opening of the permeability transition pore, allowing unregulated production of oxysterols via CYP27A1, allowing the accumulation of esterified forms of this oxysterol within human atherosclerotic lesions. Defective cholesterol efflux also induces endoplasmic reticulum stress, proteasomal degradation of ABCA1 and Fas-dependent apoptosis, replicating findings in macrophages in advanced atherosclerotic lesions. Small molecules targeted to mitochondria, capable of sustaining mitochondrial function or improving cholesterol trafficking may aid cholesterol efflux from macrophage 'foam' cells, regressing and stabilizing the atherosclerotic plaque.

Overexpression of steroidogenic acute regulatory protein increases macrophage cholesterol efflux to apolipoprotein AI.

AIMS: In this study, we investigated the impact of enhancing cholesterol delivery to mitochondrial sterol 27-hydroxylase, via steroidogenic acute regulatory protein (StAR), on the expression of genes involved in macrophage cholesterol homeostasis and efflux of cholesterol to apolipoprotein (apo) AI. METHODS AND RESULTS: Stably transfected, murine (RAW 264.7) macrophages were used to investigate the role of StAR in cholesterol homeostasis. Cellular responses were analysed using quantitative PCR, immunoblotting, and an LXRE reporter plasmid; [3H]cholesterol efflux was measured in the presence or absence of apoAI. Macrophage overexpression of mitochondrial cholesterol trafficking protein, StAR, activates and induces expression of liver X receptors (LXRs), and significantly alters expression of genes involved in cholesterol homeostasis, decreasing Fdps, Hmgcr, Mvk, Ldlr, and Scap, and markedly increasing Abca1 mRNA and protein. Overexpression of StAR, but not mutated 'loss-of-function' (R181L) StAR, enhanced efflux of [3H]cholesterol to apoAI, and this effect was maintained in macrophages pretreated with LDL or acetylated LDL. The effect of StAR overexpression on apoAI-dependent [3H]cholesterol efflux was mimicked by non-sterol agonist, T901317, and 27-hydroxycholesterol, and blocked by LXR inhibitor, geranylgeranyl pyrophosphate, sterol 27-hydroxylase inhibitor, GW273297x, and probucol, inhibitor of ATP binding cassette transporter A1 (ABCA1). Importantly, all observed effects of StAR overexpression were dependent upon cyclic AMP (cAMP analogue, dibutyryl cAMP), which is required for the full activity of the StAR protein to be manifested. CONCLUSION: Macrophage overexpression of StAR significantly enhances LXR-dependent apoAI- and ABCA1-dependent cholesterol efflux, by which disposal of excess arterial cholesterol deposits and atheroma regression can be achieved.

Resistin induces lipolysis and re-esterification of triacylglycerol stores, and increases cholesteryl ester deposition, in human macrophages.

Human resistin, found within atheroma, exerts inflammatory, angiogenic and proliferative effects in vascular cells and may predict coronary events. Here, we investigate mechanisms by which resistin contributes to macrophage 'foam cell' formation. Increases in macrophage (THP-1) cholesteryl ester mass, in the presence or absence of oxidized LDL, were not explained by altered cholesterol efflux. Instead, resistin enhanced fractional turnover of the endogenous triacylglycerol pool, increased uptake and decreased oxidation of exogenous fatty acids, and decreased phosphorylation of acetyl CoA carboxylase, all factors increasing the availability of fatty acyl CoA substrate for acyl CoA: cholesterol acyltransferase-1, thereby enhancing macrophage cholesteryl ester deposition.

Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis.

Strains of Bacillus cereus can produce a heat-stable toxin (cereulide). In this study, 101 Bacillus strains representing 7 Bacillus species were tested for production of heat-stable toxins. Strains of B. megaterium, B. firmus and B. simplex were found to produce novel heat-stable toxins, which showed varying levels of toxicity. B. cereus strains (18 out of 54) were positive for toxin production. Thirteen were of serovar H1, and it was of interest that some were of clinical origin. Two were of serovars 17B and 20, which are not usually implicated in the emetic syndrome. Partial purification of the novel B. megaterium, B. simplex and B. firmus toxins showed they had similar physical characteristics to the B. cereus emetic toxin, cereulide.