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; [ 3 H]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 [ 3 H]cholesterol to apoAI, and this effect was maintained in macrophages pretreated with LDL or acetylated LDL. The effect of StAR overexpression on apoAI-dependent [ 3 H]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.

Edited by Laszlo NagyKeywords: Atherosclerosis Macrophage 'Foam cell' Steroidogenic acute regulatory protein (StAR/STARD1) STARD3/metastatic lymph node 64 (MLN64) Liver X receptor Peroxisome proliferator activated receptor Retinoic acid X receptor Sterol regulatory element binding protein a b s t r a c tThe STARD1 subfamily of 'START' lipid trafficking proteins can reduce macrophage lipid content and inflammatory status (STARD1; StAR), and traffic cholesterol from endosomes (STARD3/MLN64). During macrophage differentiation, STARD1 mRNA and protein increase with sterol content, while the reverse is true for STARD3. Sterol depletion (methyl beta-cyclodextrin) enhances STARD3, and represses STARD1 expression. Agonists of Liver X receptors, peroxisome proliferator activated receptor-gamma and retinoic acid X receptors increase STARD1 expression, while hypocholesterolaemic agent, LY295427, reveals both STARD1 and STARD3 as putative SREBP-target genes. Pathophysiological 'foam cell' formation, induced by acetylated or oxidized LDL, significantly reduced both STARD1 and STARD3 gene expression. Differential regulation of STARD1 and D3 reflects their distinct roles in macrophage cholesterol metabolism, and may inform anti-atherogenic strategies.

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.

Dysregulated macrophage cholesterol homoeostasis lies at the heart of early and developing atheroma, and removal of excess cholesterol from macrophage foam cells, by efficient transport mechanisms, is central to stabilization and regression of atherosclerotic lesions. The present study demonstrates that transient overexpression of STARD3 {START [StAR (steroidogenic acute regulatory protein)-related lipid transfer] domain 3; also known as MLN64 (metastatic lymph node 64)}, an endosomal cholesterol transporter and member of the ‘START’ family of lipid trafficking proteins, induces significant increases in macrophage ABCA1 (ATP-binding cassette transporter A1) mRNA and protein, enhances [3H]cholesterol efflux to apo (apolipoprotein) AI, and reduces biosynthesis of cholesterol, cholesteryl ester, fatty acids, triacylglycerol and phospholipids from [14C]acetate, compared with controls. Notably, overexpression of STARD3 prevents increases in cholesterol esterification in response to acetylated LDL (low-density lipoprotein), blocking cholesteryl ester deposition. Thus enhanced endosomal trafficking via STARD3 induces an anti-atherogenic macrophage lipid phenotype, positing a potentially therapeutic strategy.

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.