Current Pharmaceutical Biotechnology - Volume 13, Issue 2, 2012

Treatment of lipid disorders commonly involves the use of cholesterol- and triglyceride lowering drugs, in particular statins and fibrates. Anion exchange resins, ezitimibe and niacin have also shown potential to reduce plasma lipid levels. Most treatments aim to reduce Low Density Lipoprotein (LDL) cholesterol and/or triglyceride-rich Very Low Density Lipopotein (VLDL) levels, and are often associated with an increase in cardioprotective High Density Lipoprotein (HDL). In fact, numerous epidemiological studies confirmed an inverse relationship of HDL, or a direct association of LDL to HDL ratio, and risk for cardiovascular disease. In particular the pharmacological induction of key molecules upregulating HDL levels or increasing HDL-induced reverse cholesterol transport from the periphery to the liver for bile secretion are promising therapeutic approaches to treat cardiovascular disease, but also diabetes and metabolic syndrome. However, despite cardioprotective effects of HDL being well documented, pharmaceutical approaches specifically targeting the anti-atherosclerotic activities of HDL are far from being fully understood or well developed. In addition, despite great advances in the field over recent years, the regulation of the key molecules controlling the removal of excess cholesterol in atherosclerotic plaque or triglycerides in fatty liver disease is still not fully understood. Several articles in this special issue aim to summarize important aspects in cholesterol and lipid transport that provide opportunity to ameliorate or reverse the formation of lipid-loaded foam cells in atherosclerosis or the accumulation of triglycerides in other diseases associated with lipid disorders, such as the metabolic syndrome. I. Zanooti et al. (in this issue) summarize current knowledge on the intracellular trafficking routes of cholesterol that influence cholesterol export (efflux) from cells, with a special emphasis on the contribution of ATP Binding Cassette A1 and G1 Transporters (ABCA1, ABCG1) and Scavenger Receptor Class B Type I (SR-BI). In particular, this review provides useful information on the currently available biochemical methodologies to examine lipid efflux from cells and the general considerations that have to be taken into account when addressing cholesterol efflux in cell culture. C. Buechler and S. Bauer (in this issue) address another aspect of lipid efflux relevant for pharmaceutical intervention against metabolic syndrome and atherosclerosis and summarize the growing list of proteins associated with ABCA1. Several of those are potentially involved in ABCA1 degradation, the prevention of which is a highly valuable target for drug development. Despite the large amount of research that has been published in the last decades to address the link between signal transduction and reverse cholesterol transport, concise reviews aiming to summarize the opportunities to enhance HDL and apolipoprotein AI (apoAI) -dependent reverse cholesterol transport are still missing. V. Mulay et al. (in this issue) provide a detailed description of the plethora of signaling cascades that promote atheroprotective cell behaviour, in particular the involvement of protein kinases and GTPases in the regulation of ABC transporters and SR-BI to promote removal of excess cholesterol from lipid-loaded macrophages. The potential of pharmaceutical intervention to modulate the activity of signaling proteins, thereby ameliorating atherosclerosis via increasing the activity of cholesterol transporters, is discussed. Despite the major advances in the understanding of cellular cholesterol transport, the knowledge of inter-compartment sterol trafficking is still limited, in part due to the experimental tools yet being insufficient to study cholesterol transport with high accuracy without simultaneously perturbing the experimental system. M. Traini and W. Jessup (in this issue) summarize the advantages of combining genomic strategies, imaging techniques and biochemistry in model organisms such as yeast and drosophila to complete missing links in intracellular sterol transport, lipid storage and metabolism. In addition, the exciting opportunities of functional genomics, but also the limitations in validating identified candidates using RNAi libraries, automated microscopy and visualization of sterols in living cells are discussed. D. Wüstner (in this issue) gives an overview of the challenges and the limitations of most experimental tools aiming to visualize and measure sterol transport and organelle cholesterol content. In addition, recent technical advances in the field to visualize and quantitate cholesterol dynamics using fluorescent analogues are described in detail. Alternatively, to study the trafficking of HDL-derived lipids in cells, C. Roehrl et al. (in this issue) describe the combined use of light and electron microscopy to follow the cellular fate of fluorescent cholesterol analogues incorporated into HDL particles. Using diaminobenzidine photooxidation to convert the fluorescent signal into electron-dense particles for electron microscopical inspection, the authors show that reconstituted HDL particles labeled with BODIPYcholesterol or BODIPY-cholesteryl oleate are an appropriate tool to investigate compartmental distribution of sterols at high resolution. Finally, two articles provide pharmaceutically and clinically relevant aspects of cholesterol and triglyceride transport that have as yet received limited attention in the field of lipoprotein research. J. Heeren and O. Bruns (in this issue) summarize the potential of nanoparticles-based imaging technologies for the non-invasive assessment of atherosclerotic lesions. Indeed, the properties of nanoparticles might offer future opportunities for quantitative measurements of lipoprotein metabolism in patients. Ultimately, nanoparticles could potentially be used to non-invasively evaluate foam cell formation, the early hallmark of atherosclerosis, lesion progression as well as regression in response to therapeutic intervention. Alternatively, nanoparticles could also be useful as carrier systems for anti-atherosclerotic drugs. In addition, M.S. Kim et al (in this issue) summarize the potential of natural medicines for the treatment of non-alcoholic fatty liver disease (NAFLD). Lipid accumulation during the development of NAFLD is characterized by the formation of triglyceride-rich lipid droplets. Given that active compounds within natural herbs that lower hepatic triglycerides are poorly characterized, medium-high throughput screening assays could provide opportunities for lead identification and drug development. The analysis of protein markers that can monitor the magnitude of LD formation have become attractive candidates to assess the efficacy of drugs aiming to reduce hepatic lipid accumulation. Additionally, the authors provide several examples that the quantification of the size and number of lipid droplets could also be developed into an automated microscopy based assay to screen compound libraries derived from natural medicines for their potential to reduce NAFLD.....

Non-alcoholic fatty liver disease (NAFLD) is emerging as a prominent condition in Western countries. In this review we describe the characteristics and current treatments of NAFLD and discuss opportunities for developing new therapeutic management approaches, with a particular emphasis on development of animal studies and in vitro assays for identification of components of natural product medicines. The main manifestation of NAFLD is hepatic lipid accumulation in the form of lipid droplets (LDs), known as hepatic steatosis (fatty liver). Current treatments for NAFLD generally aim to reduce triglyceride (TG) accumulation, often utilizing thiazolidinedines (TZDs) and fibrates, which are known to lower TG levels in hyperlipidemia, diabetes and metabolic syndrome. Both of these compounds act through activation of nuclear receptors of the Peroxisome Proliferator-Activated Receptor (PPAR) family, thereby activating genes involved in triglyceride metabolism. Thus treatment using natural PPAR and PPAR ligands, such as polyunsaturated fatty acids (PUFA), has also been considered. Alternatively, natural medicines for the treatment of NAFLD have a long and successful history of controlling disease without prominent side effects. However, active compounds in natural medicine responsible for lowering hepatic TG levels are yet poorly characterized. This points to the need for medium-high throughput screening assays to identify active components within natural herbs. As outlined in this review, the quantification of the size and number of lipid droplets could provide an opportunity to screen compound libraries derived from natural medicine for their potential to reduce NAFLD.

Intracellular lipid homeostasis is regulated by multiple mechanisms devoted to the tight control of cholesterol levels. Cholesterol efflux to extracellular acceptors represents a cellular response to excess accumulation of lipids that occurs by both passive and active processes and was shown to exert a beneficial, antiatherosclerotic activity. Up to now 3 lipid transporters responsible for cholesterol removal from cells have been characterized: ATP Binding Cassette A1 (ABCA1), ATP Binding Cassette G1 (ABCG1) and Scavenger Receptor Class B Type I (SR-BI). These proteins widely differ in the pathway of efflux they mediate, as well as in the nature of extracellular acceptors they interact with. The experimental investigation of cholesterol efflux pathways can be efficiently performed in vitro, following precise criteria in the selection of cell types and extracellular acceptors to specifically investigate the mechanism involved. The aim of this review is to describe how lipid transporter-mediated cholesterol efflux influences the intracellular trafficking of cholesterol and to provide methodological considerations for experimental evaluation of this process.

Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow the cellular movement of this essential lipid molecule. In this article, a survey of the various methods being used for analysis of sterol trafficking is given. Various classical biochemical methods are presented and their suitability for analysis of sterol trafficking is assessed. Special emphasis is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented.

The metabolic syndrome is defined as a cluster of disorders including visceral obesity, insulin resistance, hypertension, hypertriglyceridemia and low HDL. Patients have an increased risk to develop atherosclerotic disease characterized by excessive macrophage cholesterol deposition in the vascular wall. HDL removes excess cellular cholesterol which is subsequently transported to the liver for biliary excretion and thereby preserves cholesterol homeostasis. Circulating HDL levels are maintained by hepatic ATP-binding cassette transporter A1 (ABCA1) which also transports peripheral cell cholesterol to extracellular lipid acceptors. Lipid export activity of ABCA1 improves pancreatic -cell function and ameliorates insulin release. ABCA1 affects plasma membrane cholesterol distribution and formation of lipid rafts representing signalling platforms for diverse receptors including toll-like receptor 4 (TLR4). Pharmacological activation of ABCA1 pathways presumably progresses metabolic diseases, and current approaches demonstrate beneficial effects of small peptides mimicking ABCA1 ligands which stabilize ABCA1 and enhance lipid efflux similar to its physiological acceptor apolipoprotein A-I. Research of the last decade has resulted in the identification of several ABCA1 binding proteins influencing ABCA1 signalling, stability and activity. In the current review the proteins suggested to form a complex with ABCA1 are summarized and their up to now characterized features towards ABCA1 functions are described.

Diaminobenzidine (DAB) photooxidation is a method for conversion of fluorescent signals into electron-dense precipitates that are visible in the electron microscope. Recently, we have applied this method to analyze organelles involved in holo-high density lipoprotein (HDL) particle uptake at the ultrastructural level. In the present work we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids in HepG2 cells. By the combined light-electron microscopic method and with the aid of the DAB photooxidation technique, it became possible for the first time to visualize different intracellular pathways of lipoprotein particle-derived lipids and analyze the compartments involved at the ultrastructural level. HDL-Alexa 568 was used to visualize holo-HDL particle uptake. Reconstituted HDL particles containing the fluorescent cholesterol analogues Bodipy-cholesterol, Bodipy-cholesteryl oleate, or cholesteryl Bodipy-ester were used to visualize uptake of the HDL-associated sterol. In Bodipy-cholesteryl oleate and cholesteryl Bodipy-ester, the cholesterol moiety or the fatty acid moiety is fluorescently labeled, respectively; in contrast, Bodipy-cholesterol is an analogue of free cholesterol. The cellular compartments involved in their intracellular routes after uptake were analyzed in the fluorescence and electron microscope after DAB photooxidation. Bodipy-cholesterol was found to be localized in tubular endosomes and multivesicular bodies (MVBs), in the trans-Golgi network, and in stacked Golgi cisternae. In contrast, HepG2 cells incubated with HDL containing Bodipy-cholesteryl oleate or cholesteryl Bodipyester gave an uptake pattern comparable to that of holo-HDL particles, with MVBs being involved. Bodipy-cholesteryl oleate was also found in lysosomes. These results indicate that HDL-derived cholesterol and cholesteryl ester are transported by different intracellular pathways in HepG2 cells. Thus, the DAB photooxidation method enables the analysis of intracellular transport of lipoprotein particle-derived lipids at the light and at the ultrastructural level.

The growing prevalence of disorders of lipid and sterol homeostasis such as obesity and atherosclerosis in Western societies has increased demand for the development of therapies for their prevention and treatment. Crucial for this development is an understanding of the underlying cellular mechanisms which are involved in both healthy and diseased states. However, gaps remain in our knowledge of fundamental processes, such as intracellular sterol transport, lipid storage and mobilisation. Functional genomic strategies, such as genome-wide gene knockdown screens, are increasingly being exploited as powerful strategies to fill these gaps. This review discusses experimental approaches and findings in recent functional genomics studies of sterol and lipid biology, both in model organisms and human cells.

Binding of High Density Lipoprotein (HDL) and its major apolipoprotein A-I (apoA-I) to cell surface receptors is believed to initiate a plethora of signaling cascades that promote atheroprotective cell behavior, including the removal of excess cholesterol from lipid-loaded macrophages. More specifically, HDL and apoA-I binding to scavenger receptor BI (SR-BI) and ATP-binding cassette (ABC) transporter A1 has been shown to activate protein kinase A and C (PKA, PKC), Rac/Rho GTPases, Janus Kinase 2 (JAK2), calmodulin as well as mitogen-activated protein kinases (MAPK). Some of these signaling events upregulate mobilization of cholesterol from cellular pools, while others promote efflux pathways through increased expression, stability, and cell surface localization of SR-BI and ABCA1. This review aims to summarize the current knowledge of HDL- and apoA-I -induced signal transduction pathways that are linked to cholesterol efflux and discusses the underlying mechanisms that could couple ligand binding to SR-BI and ABCA1 with signaling and cholesterol export. Additional focus is given on the potential of pharmacological intervention to modulate the activity of signaling cascades for the inhibition or regression of cholesterol accumulation in atherosclerotic lesions.

Nanotechnology deals with structures with a maximum size of 100 nanometers and is applied in various scientific disciplines. The basis for this is its potential to create many new materials such as nanoparticles which are suitable for a vast range of applications in electronics or energy production but also in biomedicine. Nanoparticles have exceptional physical properties useful for different applications ranging from material sciences to biomedical imaging. In life sciences nanoparticles provide a novel tool to study metabolic processes such as the metabolism of lipoproteins or to noninvasively detect diseases in a very early stage. Major hallmarks of early atherosclerotic lesion formation are endothelial dysfunction and accumulation of large amounts of lipoprotein-derived cholesterol esters in macrophages within the vessel wall. Since conventional methods such as plasma marker analyses are not specific and sensitive enough to reliably assess the risk of cardiovascular events at an early stage, nanoparticles-based imaging technologies might provide a valuable tool for the non-invasive assessment of atherosclerotic lesions in the future. In this review, we will give an overview on the characteristics of modern nanoparticles and will emphasize the current studies utilizing nanoparticles for the visualization of both lipoprotein metabolism and atherosclerosis.

Detection of prostate carcinoma metastases is currently performed either via indirect tests like the prostate specific antigen (PSA) or prostate cancer gene 3 (PCA3) or by biopsies from masses found with medical imaging methods. Our goal was to use an ectopic odorant receptor to target prostate-derived cells throughout the body for imaging by magnetic resonance and fluorescence imaging. We synthesized a conjugate containing undecylic aldehyde (an antagonist of the human olfactory receptor hOR17-4), gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (a common magnetic resonance contrast agent) and fluorescein isothiocyanate (a fluorescent dye). Two different prostate cancer cell lines as well as five other different malignant cell lines and healthy prostate epithelial cells were incubated with this conjugate and evaluated by flow cytometry, confocal laser scanning microscopy and magnetic resonance imaging. The prostate- derived healthy and malignant cells showed stronger fluorescence than the non-prostate cancer cell lines in the flow cytometry and confocal laser scanning microscopy experiments. In the magnetic resonance imaging experiments the T1 relaxation time reduction (higher signal intensity) was also stronger for the prostate-derived cells than for the non-prostate cells. The examined conjugate showed high prostate-cell-specificity. This property makes it of potential value in the diagnosis of prostate cancer lymph node metastases.