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.

In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound closure rates and hemichannel signalling in adult keratinocytes (AK) and fibroblasts sourced from juvenile foreskin (JFF), human neonatal fibroblasts (HNDF) and adult dermal tissue (ADF). The impact of these agents, following 24 h exposure, on GJA1 (encoding Connexin43), Ki67 and TGF-β1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100–100 μM) attenuated hemichannel activity. In AK and JFF cells, Gap27 (100 nM–100 μM) enhanced scrape wound closure rates by ~50% but did not influence movement in HNDF or ADF cells. In both JF and AK cells, exposure to Gap27 for 24 h reduced the level of Cx43 protein expression but did not affect the level in ADF and HNDF cells. Connexin43-SiRNA enhanced scrape wound closure in all the cell types under investigation. In HDNF and ADF, Connexin43-SiRNA enhanced cell proliferation rates, with enhanced proliferation also observed following exposure of HDNF to Gap27. By contrast, in JFF and AK cells no changes in proliferation occurred. In JFF cells, Connexin43-SiRNA enhanced TGF-β1 levels and in JFF and ADF cells both Connexin43-SiRNA and Gap27 enhanced pSmad3 protein expression levels. We conclude that Connexin43 signalling plays an important role in cell migration in keratinocytes and foreskin derived fibroblasts, however, different pathways are evoked and in dermal derived adult and neonatal fibroblasts, inhibition of Connexin43 signalling plays a more significant role in regulating cell proliferation than cell migration.

Effective cholesterol homoeostasis is essential in maintaining cellular function, and this is achieved by a network of lipid-responsive nuclear transcription factors, and enzymes, receptors and transporters subject to post-transcriptional and post-translational regulation, whereas loss of these elegant, tightly regulated homoeostatic responses is integral to disease pathologies. Recent data suggest that sterol-binding sensors, exchangers and transporters contribute to regulation of cellular cholesterol homoeostasis and that genetic overexpression or deletion, or mutations, in a number of these proteins are linked with diseases, including atherosclerosis, dyslipidaemia, diabetes, congenital lipoid adrenal hyperplasia, cancer, autosomal dominant hearing loss and male infertility. This review focuses on current evidence exploring the function of members of the ‘START’ (steroidogenic acute regulatory protein-related lipid transfer) and ‘ORP’ (oxysterol-binding protein-related proteins) families of sterol-binding proteins in sterol homoeostasis in eukaryotic cells, and the evidence that they represent valid therapeutic targets to alleviate human disease.

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.

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.