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.

The aim of this review is to explore the role of mitochondria in regulating macrophage sterol homeostasis and inflammatory responses within the aetiology of atherosclerosis. Macrophage generation of oxysterol activators of liver X receptors (LXRs), via sterol 27-hydroxylase, is regulated by the rate of flux of cholesterol to the inner mitochondrial membrane, via a complex of cholesterol trafficking proteins. Oxysterols are key signalling molecules, regulating the transcriptional activity of LXRs which coordinate macrophage sterol metabolism and cytokine production, key features influencing the impact of these cells within atherosclerotic lesions. The precise identity of the complex of proteins mediating mitochondrial cholesterol trafficking in macrophages remains a matter of debate, but may include steroidogenic acute regulatory protein and translocator protein. There is clear evidence that targeting either of these proteins enhances removal of cholesterol via LXRα-dependent induction of ATP binding cassette transporters (ABCA1, ABCG1) and limits the production of inflammatory cytokines; interventions which influence mitochondrial structure and bioenergetics also impact on removal of cholesterol from macrophages. Thus, molecules which can sustain or improve mitochondrial structure, the function of the electron transport chain, or increase the activity of components of the protein complex involved in cholesterol transfer, may therefore have utility in limiting or regressing atheroma development, reducing the incidence of coronary heart disease and myocardial infarction.

The functional relationship between cardioacceleratory peptide 2 (CAP2) and hindgut activity during wandering behaviour was investigated in fifth-instar larvae of the tobacco hawkmoth Manduca sexta. Inspection of the alimentary canal on the day prior to wandering showed that the gut, in preparation for metamorphosis, was voided of all contents by 18:00 h. Associated with this event, which we refer to as ‘gut emptying’, was an increase in the frequency of hindgut contractions measured in vivo. No change in heart activity was seen during this developmental period. Measurements of the amount of CAP2 in the central nervous system (CNS) of fifth-instar caterpillars revealed that CAP2 storage levels declined sharply on the day of gut emptying. The drop in CNS levels of CAP2 at gut emptying was temporally correlated with the appearance of CAP2 in the haemolymph. CAP2, when applied at physiological concentrations to an in vitro larval hindgut bioassay, caused changes in several parameters, including contraction frequency and amplitude, and basal tension. In vivo administration of CAP2 elicited hindgut responses that were qualitatively and quantitatively similar to those seen in vitro. Developmental studies on changes in CAP2 responsiveness during the last larval instar demonstrated that the hindgut is maximally sensitive to CAP2 on the day of gut emptying. Direct evidence in support of a role for CAP2 in fifth-instar larvae was provided by experiments in which the increase in gut activity in vivo seen at gut emptying was significantly reduced by injections of an anti-CAP antibody. Based on data from cobalt backfills and anti-CAP immunohistochemical staining, we propose that CAP2 exerts its effect on the larval hindgut at wandering via a local release from CAP-containing neurones in the terminal ganglion that project directly to the hindgut.

The purpose of this study was to investigate, through the analysis of word recognition errors, the relationship between conceptual tempo and developmental changes in the bases for memory organization (phonetic or semantic). Following one presentation of a 69-item study list 96 reflective and impulsive third- and sixth-grade boys and girls were presented a recognition test list which contained words that were semantically or phonetically related to words which had appeared in the study list. Neither conceptual tempo nor age was clearly related to the number or type of recognition errors made. It is suggested that the previously demonstrated superiority of reflective, relative to impulsive, feature-analytic processes may be restricted to conditions of visual presentation.