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The role of membrane cholesterol in modulating G protein-coupled receptor (GPCR) structure and function has emerged as a powerful theme in contemporary biology. In this paper, we report the subtlety and stringency involved in the interaction of sterols with the serotonin1A receptor. For this, we utilized two immediate biosynthetic precursors of cholesterol, 7-dehydrocholesterol (7-DHC) and desmosterol, which differ with cholesterol merely in a double bond in their chemical structures in a position-dependent manner. We show that whereas 7-DHC could not support the ligand binding function of the serotonin1A receptor in live cells, desmosterol could partially support it. Importantly, depletion and enrichment of membrane cholesterol over basal level resulted in an increase and reduction of the basal receptor activity, respectively. These results demonstrate the relevance of optimal membrane cholesterol in maintaining the activity of the serotonin1A receptor, thereby elucidating the relevance of cellular cholesterol homeostasis.

Over the years protein interaction and prediction of membrane protein have been a pivotal research area for all researchers. For both prokaryotes and eukaryotes Adenosine Triphosphate-(ATP) binding cassette (ABC) genes plays a significant role. In our analysis, we concentrate on human part of ABC genes. In case of living organisms transport of precise molecules across lipid membranes has been treated as vital part and for that reason a bigger transporter is required to carry out the molecules. Here ABC transporter families are evolved to transport the specific molecules such as sugars, amino acid, peptides, proteins, ions etc. within the plasma membrane. As we know another important component of human being is cholesterol, which is a major component in cell membrane and its main functions are to maintain integrity and mechanical stability. Each and every time, membrane cholesterolsareinteracted with membrane protein in both N-C terminuses and target valid sequence(s) which has relevance in human diseases. In this manuscript we have applied Fuzzy C-Means (FCM) with Support Vector Machine (SVM) algorithm for prediction of cellular cholesterol with ABC genes. Our experiments have been performed well using ABCdata set.

The influence of the cellular cholesterol content on the cytotoxicity of endovanilloids acyldopamines was studied in MDA-MB-231 and MCF 10A cells. The activity of acyldopamines depends on the cellular cholesterol content, and a decrease in cholesterol content increases the cytotoxicity of acyldopamines.

Despite the significant advancements made in targeted anti-cancer therapy, drug resistance constitutes a multifaceted phenomenon leading to therapy failure and ultimately mortality. Emerging experimental evidence highlight the role of cholesterol metabolism in facilitating drug resistance in cancer. This review aims to describe the role of cholesterol and its downstream metabolites in facilitating multi-drug resistance in cancer. We focus on specific signaling pathways that contribute to drug resistance and the link between these pathways and cholesterol. Additionally, we briefly discuss the molecular mechanisms related to the epithelial-mesenchymal transition (EMT), and the documented link between EMT, metastasis, and drug resistance. We illustrate this by specifically focusing on hypoxia and the role hypoxia plays in influencing cellular cholesterol content following EMT induction. Finally, we also provide preliminary findings delineating the crucial role of cholesterol in EMT and discuss whether targeting cholesterol could serve as a novel means of combatting drug resistance in cancer progression and metastasis.

Cell classification is a fundamental task in biological research and medical practices. In this study, we proposed a singlecell classification pipeline through machine learning and hyperspectral stimulated Raman scattering imaging. The pipeline proposed is validated by using hyperspectral SRS images of two types of pancreatic cancer cells before and after the treatment of drugs that affects cellular cholesterol level. The result demonstrates that the proposed machine learning pipeline is capable of classifying cells with different metabolite dynamics, which provides possibilities for wide applications in cell analysis.

In specialized cell types, lysosome-related organelles support regulated secretory pathways, whereas in nonspecialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol-storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in nonspecialized cells and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel mucolipin-1, as well as FYCO1, the AP1 adaptor, and its partner Gadkin. We conclude that endo-lysosomes in nonspecialized cells can acquire secretory functions elicited by cyclodextrin and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

Cholesterol is an essential component of eukaryotic cellular membranes. Information about its subcellular localization and transport pathways inside cells are key for the understanding and treatment of cholesterol-related diseases. In this review we give an overview over the most commonly used methods that contributed to our current understanding of subcellular cholesterol localization and transport routes. First, we discuss methods that provide insights into cholesterol metabolism based on readouts of downstream effects such as esterification. Subsequently, we focus on the use of cholesterol-binding molecules as probes that facilitate visualization and quantification of sterols inside of cells. Finally, we explore different analogues of cholesterol which, when taken up by living cells, are integrated and transported in a similar fashion as endogenous sterols. Taken together, we highlight the challenges and advantages of each method such that researchers studying aspects of cholesterol transport may choose the most pertinent approach for their problem.

Cholesterol has gained tremendous attention as an essential lipid in the life cycle of virtually all viruses. These seem to have developed manifold strategies to modulate the cholesterol metabolism to the side of lipid uptake and de novo synthesis. In turn, affecting the cholesterol homeostasis has emerged as novel broad-spectrum antiviral strategy. On the other hand, the innate immune system is similarly regulated by the lipid and stimulated by its derivatives. This certainly requires attention in the design of antiviral strategies aiming to decrease cellular cholesterol, as evidence accumulates that withdrawal of cholesterol hampers innate immunity. Secondly, there are exceptions to the rule of the abovementioned virus-induced metabolic shift toward cholesterol anabolism. It therefore is of interest to dissect underlying regulatory mechanisms, which we aimed for in this minireview. We further collected evidence for intracellular cholesterol concentrations being less important in viral life cycles as compared to the spatial distribution of the lipid. Various routes of cholesterol trafficking were found to be hijacked in viral infections with respect to organelle-endosome contact sites mediating cholesterol shuttling. Thus, re-distribution of cellular cholesterol in the context of viral infections requires more attention in ongoing research. As a final aim, a pan-antiviral treatment could be found just within the transport and re-adjustment of local cholesterol concentrations. Thus, we aimed to emphasize the importance of the regulatory roles the endosomal system fulfils herein and hope to stimulate research in this field.

High-density lipoprotein cholesterol (HDL) is the major promoter of reverse cholesterol transport and efflux of excess cellular cholesterol. The functions of HDL, such as cholesterol efflux, are associated with cardiovascular disease rather than HDL levels. We have reviewed the evidence base on the major classes of phytochemicals, including polyphenols, alkaloids, carotenoids, phytosterols, and fatty acids, and their effects on macrophage cholesterol efflux and its major pathways. Phytochemicals show the potential to improve the efficiency of each of these pathways. The findings are mainly in preclinical studies, and more clinical research is warranted in this area to develop novel clinical applications.

The inverse relationship between low plasma high-density lipoprotein cholesterol (HDL-C) concentrations and increased risk of Atherosclerotic Cardiovascular Disease (ASCVD) is well-known. However, plasma HDL-C concentrations are highly variable in subjects with ASCVD. In clinical outcome trials, pharmacotherapies that increase HDL-C concentrations are not associated with a reduction in ASCVD events. A causal relationship between HDL-C and ASCVD has also been questioned by Mendelian randomization studies and genome-wide association studies of genetic variants associated with plasma HDL-C concentrations. The U-shaped association between plasma HDL-C concentrations and mortality observed in several epidemiological studies implicates both low and very high plasma HDL-C concentrations in the etiology of ASCVD and non- ASCVD mortality. These data do not collectively support a causal association between HDL-C and ASCVD risk. Therefore, the hypothesis concerning the association between HDL and ASCVD has shifted from focus on plasma concentrations to the concept of functionality, in particular cellular cholesterol efflux and HDL holoparticle transport. In this review, we focus on these new concepts and provide a new framework for understanding and testing the role of HDL in ASCVD.

Context Severe hypothyroidism has profound effects on lipoprotein metabolism including high-density lipoprotein (HDL) cholesterol elevations but effects on HDL function metrics are unknown. Objective To determine the impact of severe short-term hypothyroidism on HDL particle characteristics, HDL cholesterol efflux capacity (CEC), and HDL antioxidative capacity. Design Observational study with variables measured during severe short-term hypothyroidism (median TSH 81 mU/L) and after 20 weeks of thyroid hormone supplementation (median TSH 0.03 mU/L) (Netherlands Trial Registry ID 7228). Setting University hospital setting in The Netherlands. Patients Seventeen patients who had undergone a total thyroidectomy for differentiated thyroid carcinoma. Main outcome measures HDL particle characteristics (nuclear magnetic resonance spectrometry), CEC (human THP-1-derived macrophage foam cells and apolipoprotein B-depleted plasma), and HDL anti-oxidative capacity (inhibition of low-density lipoprotein oxidation). Results During hypothyroidism plasma total cholesterol, HDL cholesterol and apolipoprotein A-I were increased (P ≤ 0.001). HDL particle concentration was unchanged, but there was a shift in HDL subclasses toward larger HDL particles (P < 0.001). CEC was decreased (P = 0.035), also when corrected for HDL cholesterol (P < 0.001) or HDL particle concentration (P = 0.011). HDL antioxidative capacity did not change. Conclusion During severe short-term hypothyroidism CEC, an important antiatherogenic metric of HDL function, is impaired. HDL cholesterol and larger HDL particles are increased but HDL particle concentration is unchanged. Combined, these findings suggest that HDL quality and quantity are not improved, reflecting dysfunctional HDL in hypothyroidism.

Influenza A virus (IAV) is a common pathogen of respiratory disease. The IAV-induced seasonal epidemics and the sporadic pandemics are associated with high morbidity and mortality. Therefore, effective protection and therapy for IAV infections is an important challenge in countering this public health threat. Because vaccinations only protect against known circulating strains, and the currently available antivirals pose the risk of resistance formation, drugs targeting host cell factors needed for viral replication offer a promising therapeutic approach. In this study, we describe the use of the antifungal therapeutics posaconazole and itraconazole in the therapy of IAV. We show that both drugs efficiently inhibit the propagation of IAV in the cell culture model without being cytotoxic. The mode of action is probably based on several targets and includes both a priming of the interferon response and the induced imbalance of cellular cholesterol. The antiviral effect of itraconazole could be confirmed in the mouse model, where the administration of itraconazole led to a drastic reduction in mortality and a significant increase in the survival rate. Thus, our data indicate a promising therapeutic potential of at least itraconazole in influenza therapy.

Supplemental Digital Content is available in the text., Objective— Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology. Approach and Results— Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. Conclusions— To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.

We formulate, parameterise and analyse a mathematical model of the mevalonate pathway, a key pathway in the synthesis of cholesterol. Of high clinical importance, the pathway incorporates rate limiting enzymatic reactions with multiple negative feedbacks. In this work we investigate the pathway dynamics and demonstrate that rate limiting steps and negative feedbacks within it act in concert to tightly regulate intracellular cholesterol levels. Formulated using the theory of nonlinear ordinary differential equations and parameterised in the context of a hepatocyte, the governing equations are analysed numerically and analytically. Sensitivity and mathematical analysis demonstrate the importance of the two rate limiting enzymes 3-hydroxy-3-methylglutaryl-CoA reductase and squalene synthase in controlling the concentration of substrates within the pathway as well as that of cholesterol. The role of individual feedbacks, both global (between that of cholesterol and sterol regulatory element-binding protein 2; SREBP-2) and local internal (between substrates in the pathway) are investigated. We find that whilst the cholesterol SREBP-2 feedback regulates the overall system dynamics, local feedbacks activate within the pathway to tightly regulate the overall cellular cholesterol concentration. The network stability is analysed by constructing a reduced model of the fall pathway and is shown to exhibit one real, stable steady-state. We close by addressing the biological question as to how farnesyl-PP levels are affected by CYP51 inhibition, and demonstrate that the regulatory mechanisms within the network work in unison to ensure they remain bounded.

Neuroblastoma (NB) is an extra cranial pediatric embryonal tumor most prevalent in children less than 1 year of age. NB accounts for 7% of all pediatric cancers but accounts for 15% of all childhood cancer deaths. Scavenger receptor class B type 1 (SR-B1), a mediator of cellular cholesterol uptake, is overexpressed in and have been linked to the aggressiveness of many cancers. Nevertheless, no studies have so far investigated the relationship between SR-B1 and NB. Elucidation of receptors that promote NB may pave the way for discovery of new therapeutic targets. Here we show that inhibition of SR-B1 reduced cell survival, migration and invasion, and cholesterol content in NB cell lines. Additionally analysis of SR-B1 levels in NB patient biopsies using the R2: Genomics Analysis and Visualization Platform showed that high SR-B1 expression correlated with decreased overall and event-free survival.

Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1I1061T, has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat.

// Raghavendra Gowda 1, 5, 6, 7 , Gajanan S. Inamdar 1 , Omer Kuzu 1 , Saketh S. Dinavahi 1 , Jacek Krzeminski 1 , Madhu Babu Battu 8 , Sreedhara R. Voleti 8 , Shantu Amin 1 , Gavin P. Robertson 1, 2, 3, 4, 5, 6, 7 1 Department of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA 2 Department of Pathology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA 3 Department of Dermatology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA 4 Department of Surgery, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA 5 The Penn State Melanoma and Skin Cancer Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA 6 Penn State Melanoma Therapeutics Program, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA 7 Foreman Foundation for Melanoma Research Laboratory, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA 8 Drug Discovery Research Laboratory, INDRAS Private Limited, Hyderabad, India 500040 Correspondence to: Gavin P. Robertson, email: gpr11@psu.edu Keywords: melanoma, leelamine, abietic acid, structure-activity relationship, AKT Received: December 23, 2016 Accepted: February 23, 2017 Published: March 08, 2017 ABSTRACT Leelamine is an anticancer chemotherapeutic agent inhibiting intracellular cholesterol transport. Cell death mediated by leelamine occurs due to the lysosomotropic property of the compound, its accumulation in the lysosome, and inhibition of cholesterol transport leading to lack of availability for key processes required for functioning of cancer cells. The present study dissects the structure-activity-relationship of leelamine using synthesized derivatives of leelamine and abietic acid, a structurally similar compound, to identify the moiety responsible for anti-cancer activity. Similar to leelamine, all active derivatives had an amino group or a similar moiety that confers a lysosomotropic property to the compound enabling its accumulation in the lysosome. Active derivatives inhibited intracellular cholesterol transport and hindered xenografted melanoma tumor development without obvious systemic toxicity. In silico studies suggested that active derivatives accumulating in lysosomes bound to NPC1, a protein responsible for cholesterol export from the lysosome, to inhibit its activity that then caused accumulation, and lack of cholesterol availability for other key cellular activities. Thus, active derivatives of leelamine or abietic acid maintained lysosomotropic properties, bound to NPC1, and disrupted cellular cholesterol transport as well as availability to retard tumor development.

Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA–based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel–dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.

Most types of cells in the body have no or very limited capacity of catabolizing cholesterol, so cholesterol efflux is essential for cholesterol homeostasis. There are multiple mechanisms responsible for cellular cholesterol efflux. Among them, the active efflux pathways are mediated primarily by the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. ABCA1 is essential for cholesterol and phospholipid efflux to apolipoprotein A-I and high density lipoprotein (HDL) biogenesis. ABCG1 promotes cholesterol efflux primarily to HDL particles. Atherosclerotic cardiovascular disease is a chronic inflammatory disease characterized by marked macrophage foam cell accumulation in atherosclerotic plaques and associated pro-inflammatory responses in lesional cells. Findings from both animal and human studies indicate a critical role of disturbed cholesterol homeostasis in pro-inflammatory responses in these cells, particularly in lesional macrophages. ABCA1 and ABCG1 are highly expressed in macrophages, particularly in response to cholesterol accumulation, and are essential in maintenance of cholesterol homeostasis. Functional deficiency of ABCA1 and ABCG1 in macrophage markedly increases atherogenesis, with exacerbated inflammatory responses. ABCA1 and ABCG1 also play a critical role in control of hematopoietic stem and progenitor cell (HSPC) proliferation and extramedullary hematopoiesis. Hematopoietic ABCA1 and ABCG1 deficiencies cause marked HSPC expansion and extramedullary hematopoiesis, particularly in hypercholesterolemia, and lead to marked monocytosis and neutrophilia with exacerbated pro-inflammatory responses. All these contribute to atherosclerosis. In this chapter, we describe these findings and discuss the current understanding of the underlying mechanisms. We also discuss other ABC transporters such as ABCG4, which also promotes cholesterol efflux to HDL and controls megakaryocyte proliferation and platelet biogenesis. By this pathway, ABCG4 also modulates atherogenesis. Therapeutic approaches targeting the pathways and mechanisms described also are discussed.

Significance Biological systems function through the interaction of numerous molecules influencing a variety of biochemical reactions. However, most biological systems are still only partially understood. This paper introduces GeneFishing, a method for “fishing out” candidate genes in a biological process. The method is “semisupervised” using a set of “bait” genes (i.e., ones previously known to be relevant to the same process). GeneFishing effectively combines modern and traditional statistical ideas for analyzing both big and small data. We applied this method to cholesterol-related genes and identified several interesting phenomena. GeneFishing has the potential for pointing to functional importance in known but poorly studied genes, and its underlying framework is broadly applicable inside and outside biology., Rapid advances in genomic technologies have led to a wealth of diverse data, from which novel discoveries can be gleaned through the application of robust statistical and computational methods. Here, we describe GeneFishing, a semisupervised computational approach to reconstruct context-specific portraits of biological processes by leveraging gene–gene coexpression information. GeneFishing incorporates multiple high-dimensional statistical ideas, including dimensionality reduction, clustering, subsampling, and results aggregation, to produce robust results. To illustrate the power of our method, we applied it using 21 genes involved in cholesterol metabolism as “bait” to “fish out” (or identify) genes not previously identified as being connected to cholesterol metabolism. Using simulation and real datasets, we found that the results obtained through GeneFishing were more interesting for our study than those provided by related gene prioritization methods. In particular, application of GeneFishing to the GTEx liver RNA sequencing (RNAseq) data not only reidentified many known cholesterol-related genes, but also pointed to glyoxalase I (GLO1) as a gene implicated in cholesterol metabolism. In a follow-up experiment, we found that GLO1 knockdown in human hepatoma cell lines increased levels of cellular cholesterol ester, validating a role for GLO1 in cholesterol metabolism. In addition, we performed pantissue analysis by applying GeneFishing on various tissues and identified many potential tissue-specific cholesterol metabolism-related genes. GeneFishing appears to be a powerful tool for identifying related components of complex biological systems and may be used across a wide range of applications.

Mammalian cells carefully control their cholesterol levels by employing multiple feedback mechanisms to regulate synthesis of cholesterol and uptake of cholesterol from circulating lipoproteins. Most of a cell's cholesterol (~80% of total) is in the plasma membrane (PM), but the protein machinery that regulates cellular cholesterol resides in the endoplasmic reticulum (ER) membrane, which contains a very small fraction (~1% of total) of a cell's cholesterol. How does the ER communicate with PM to monitor cholesterol levels in that membrane? Here, we describe a tool, ALOD4, that helps us answer this question. ALOD4 traps cholesterol at the PM, leading to depletion of ER cholesterol without altering total cell cholesterol. The effects of ALOD4 are reversible. This tool has been used to show that the ER is able to continuously sample cholesterol from PM, providing ER with information about levels of PM cholesterol.

Lipid metabolism derangement contributes to increased cardiovascular risk in Rheumatoid Arthritis (RA). It is still debated whether and how tocilizumab, an interleukin-6 receptor inhibitor used in active RA, impacts cardiovascular risk. We studied the effect of tocilizumab on the regulation of macrophage cholesterol homeostasis, measuring patient serum ability to respectively load (cholesterol loading capacity, CLC) and discharge (cholesterol efflux capacity, CEC) cells with cholesterol. Patients with RA (n = 8) were studied before and after 4 and 12 weeks of tocilizumab treatment. CLC was measured by a fluorimetric assay of intracellular cholesterol content in human macrophages and CEC was measured for the three main pathways, mediated by the transporters Scavenger Receptor class B-type I (SR-BI), ATP binding cassette-G1 (ABCG1) and -A1 (ABCA1) in specific cell models. After 12 weeks of tocilizumab treatment, serum LDL cholesterol levels were increased, while CLC was reduced. HDL cholesterol levels were unchanged, but CEC was significantly ameliorated for the SR-BI and ABCG1 pathways with respect to baseline. Tocilizumab reduces LDL pro-atherogenic potential despite increasing their serum levels and increases HDL protective activity in RA. The data of our pilot study suggest that tocilizumab regulates lipoprotein function in selected patient populations and lay the groundwork for future larger studies.

International audience; Eukaryotic cells employ distinct means to release specific signals and material. Research within the last decade has identified different types of membrane-enclosed structures collectively called extracellular vesicles (EVs) as one of them. EVs fall into two categories depending on their subcellular origin. Exosomes are generated within the endosomal system and reach the extracellular space upon fusion of multivesicular bodies. Microvesicles or microparticles are generated by shedding of the plasma membrane. Sterols are essential components of eukaryotic membranes and serve as precursors or cofactors of numerous signaling molecules; their content and subcellular distribution are tightly controlled. The prominent roles of sterols in cells raise the question of whether and how these components impact EVs. In this review, we compile evidence for cholesterol accumulation in EVs and discuss its possible contribution to their biogenesis, release, and uptake. We also consider potential implications of EVs in cellular sterol homeostasis and in cholesterol-related diseases.

Accumulation of cholesterol loaded macrophage foam cells is the hallmark of atherosclerotic lesions where high cellular cholesterol content not only contributes to the plaque volume but is also responsible for the inflammatory milieu. Causal relationship exist between macrophage cholesterol content and inflammatory status involving TLR4, NFκB or cholesterol crystal mediated activation of inflammasome and IL1β secretion. Since K+ efflux is central to inflammasome activation, in this study we examined the hypothesis that cellular cholesterol content directly affects K+ channel activity as well as K+ efflux. K+ currents were monitored in mouse peritoneal macrophages (MPMs) from chow or western diet (WD) fed LDLR-/- or LDLR-/-CEHTg mice. WD feeding led to a significant increase in the K+ currents in MPMs from LDLR-/- mice (A, red). In contrast, WD feeding did not affect K+ currents in MPMs from LDLR-/-CEHTg mice; these MPMs have reduced cellular cholesterol accumulation due to increased efflux. Consistently, cholesterol loading with AcLDL led to an increase in K+ currents in THP1 macrophages (B, red) and this increase was attenuated following cholesterol efflux. The physiological effect of changes in K+ channel activity or K+ efflux on inflammasome activation in MPMs by different activators (K+ ionophore Nigericin, N or ALUM crystals) was examined. Increase in extracellular K+ (+KCl) or inhibition of K+ efflux by glyburide significantly reduced IL1β secretion (C&D). Consistently, cellular K+ levels after LPS+N or LPS+ALUM treatment were significantly reduced (E). While in MPMs only K ATP channel inhibitors reduced IL1β secretion (F), in human THP1 macrophages inhibition of K ATP as well as Na/K pump inhibitors significantly reduced IL1β secretion (G). These data demonstrate a novel mechanism for cholesterol mediated regulation of inflammatory pathways by regulation of K+ channel activity and also illustrate species specific differences in K+ channels involved

Background and aims Research on the biologic activities of HDL, such as cholesterol efflux capacity and HDL composition, has allowed the understanding of the effect of interventions directed to improve cardiovascular risk. Previously, statin therapy has shown conflicting results about its effects on cholesterol efflux capacity of HDL; the underlying mechanisms are unclear but studies with positive effects are associated with an increase of HDL-cholesterol levels. We investigated if 10 weeks of atorvastatin therapy changes HDL efflux capacity and the chemical composition of its subpopulations. Methods In a before-after design basis, HDL-cholesterol levels, chemical composition and cholesterol efflux capacity from HDL subpopulations isolated by isophynic ultracentrifugation were assessed in plasma samples from 60 patients with type 2 diabetes mellito (T2DM) at baseline and after 10 weeks of treatment with 20 mg atorvastatin. Cholesterol efflux was measured from human THP-1 cells using large, light HDL2b and small, dense 3c subpopulations as well as total HDL as acceptors. Changes of cholesterol efflux and chemical composition of HDL after treatment were analyzed. Correlations among variables potentially involved in cholesterol efflux were evaluated. Results A significant decrease of 4% in HDL-cholesterol levels was observed from 47 (42–54) to 45 (39–56) mg/dL, p = 0.02. Cholesterol efflux from total-HDL and HDL2b and 3c subfractions was maintained unchanged after treatment. The total mass of HDL remained unaffected, except for the HDL3a subpopulation accounted for by a significant increase in total protein content. No significant correlations for variables previously known to be associated with cholesterol efflux were found in our study. Conclusions Short therapy of 10 weeks with 20 mg of atorvastatin does not modify the cholesterol efflux capacity neither the total mass of HDL2b, HDL3c and total HDL. The discrepancy with previous reports may be due to the selective effects among different classes of statins or differences in the approaches to measure cellular cholesterol efflux.

This review discusses advances in understanding how the controlled delivery of cholesterol between subcellular compartments is achieved and what novel experimental strategies are being employed to address this fundamental question. Recent work has focused on cholesterol-binding proteins that can facilitate directional cholesterol transfer between contacts of the ER and Golgi or late endosomal membranes. Increasing structural information on cholesterol-binding proteins, new modules engineered from them as well as improved imaging and gene editing techniques are providing valuable insights. There is also mounting information on how the crosstalk between cholesterol transport and nutrient signaling is orchestrated and how cellular fatty acid metabolism and cholesterol homeostasis are intertwined.