Ida Perrotta obtained her degree in Biological Sciences and a Doctorate in Animal Biology from the University of Calabria. Currently, she is Chief Technician of Transmission Electron Microscopy Unit at the Centre for Microscopy and Microanalysis (CM2), University of Calabria. She has published more than 60 scientific papers and is actively involved in research as well as in a number of international projects in the atherosclerosis field.

Introduction: Lipid droplets (LDs) are lipid-rich organelles found nearly ubiquitously in cells where they perform diverse functions such as lipid storage for energy production and membrane synthesis, intracellular trafficking, and viral replication. Long perceived as a simple reservoir of neutral lipids, LDs have recently attracted great interest in biomedical research being recognized as dynamic structures with a complex and interesting biology. In many non-adipocyte cells, LDs have been shown to be highly motile and have been often found to interact with the endoplasmic reticulum (ER), mitochondria, endosomes, and peroxisomes. To date, however, much remains unknown regarding the topographical connections between LDs and other cell organelles in the cardiovascular system, especially in atherosclerosis. In order to fill this gap, an electron microscopic analysis has been conducted on human atherosclerotic plaques and the results are presented herein.

Materials & Methods: Specimens of atherosclerotic human aorta were obtained from patients undergoing surgical repair of the aortic atherosclerotic aneurysm. Tissues were fixed in glutaraldehyde, post-fixed with osmium tetroxide, dehydrated through a graded acetone series, and embedded in araldite. Ultra-thin sections were examined with a JEOL JEM-1400 PLUS transmission electron microscope operating at 80 kV.

Findings: LDs have been found to be closely associated with the convoluted rough-surfaced ER membranes and even with the lumen of these structures probably to facilitate a non-vesicular lipid transport between the ER and the droplets. TEM examination also revealed the presence of membrane-like structures in the LD core.

Conclusion & Significance: The selective physical interaction between LDs and ER membranes in the foam cells of human atherosclerotic plaques may function as a lipid-buffer system that allows the sequestration of free fatty acid, therefore, reducing the risk of lipotoxicity. The ultrastructural anatomy of these organelles might also account for some of their specific functional properties such as the synthesis of inflammatory lipid mediators by specific membrane-spanning enzymes which might reside in their internal membranous structures.

Figure 1: Viable foam cells contain large Lipid Droplets (LDs), intact mitochondria, well-developed rER, and numerous electrondense ribosomes. LDs frequently interact with the ER (arrow) throught an extended membrane stalk.

Guido Galliani has graduated in Chemistry at Milano University in 1974 where he has spent ten years as a Research fellow and an Assistant Professor. His main research interests were physical organic chemistry, oxidation mechanism, and model reactions for oxygen reactive intermediates. He served as a R&D Director and then as a factory manager in companies producing Active Principle Ingredients (Labochim, Honeywell). Partner for ten years of a company (Chorisis) eventually sold to Euticals group, he developed API impurities characterization, design of experiments, and continuous process development. In the same years, he held an assignment as adjunct Professor at Bicocca Milano University. He has spent more than six years in Norwegian factories (Lipro, PharmaMarine), introducing new techniques and processes for omega-3 products, including technology for the industrial production of squid oil as a DHA source. Since January 2017 he is acting as a Consultant for Gleaner srls.

Until not so long ago, wax esters had an overall bad reputation. Spermaceti, jojoba oil, orange roughy oil are typical examples of wax esters, well renown insofar that they are used topically, mostly for cosmetic purposes. However, it is equally well known that these products should never be ingested, under penalty of very unpleasant consequences, such as nausea, diarrhoea, and steatorrhea. Some years ago, we started a program to understand some rather contradictory data. It is known that roe (fish eggs) is consumed as a food since time immemorial. This occurs either on roe as is (caviar, lumpfish roe) or after some treatment (Italian bottarga, Greek taramosalata, Japanese karasumi, Scandinavian Lysekils kaviar). Moreover, it was known that wax esters are an important constituent of roe, in some instances the majority component. In spite of this, no concern had ever been raised as to edibility or side effects of such products. The first results in analyzing wax esters from several roe-based foods showed that polyunsaturated fatty acids were a very important component of the acidic moiety. Successive results from other research groups confirmed our original results. These findings partially supported substantiating and launching a commercial product, Calanus® oil, extracted from copepod Calanus finmarchicus, rich in wax esters and in omega-3 polyunsaturated acids. From all this, it is clear that the term wax ester is just a rough chemical definition. Within this class of lipids different molecules are included. Some of them are essentially saturated molecules, primarily intended to assist buoyancy and to store energy. Other molecules represent a more complex metabolism, not yet fully understood. Differences are reflected in the metabolic fate when ingested. Competitive kinetics results will be presented from several wax esters and lipases, revealing suggestions about wax esters as a delivering form for omega- 3.

Jorge Maldonado-Hernández has his expertise in the development of non-invasive methods and biological markers for the diagnosis of metabolic disorders as hyperhomocysteinemia, insulin resistance and non-alcoholic fatty liver disease. He has extensive experience in the management of analytical techniques such as isotope ratio mass spectrometry, gas chromatography and liquid-mass chromatography. Recently, he has been interested in the effect of dietary lipids on the biosynthesis of ceramides in human muscle tissue.

Objective: To assess the association between plasma ceramides and hepatic steatosis (HS) in adolescents, independently of obesity.

Material & Methods: Ninety-four adolescents from two previous studies conducted and published by our crew were included. Study subjects were stratified in three groups: normal weight (n=18), obesity (n=34) and obesity + HS (n=42). The presence of HS was defined when AST/ALT ratio was <1. Ceramides subspecies (C14:0, C16:0, C18:0, C24:0 and C24:1) were determined by LC/MS.

Results: All ceramides correlated inversely with the AST/ALT ratio and directly with ALT levels in plasma; the strongest correlation was observed among C14:0 ceramide (r=-0.54 and r=0.41, respectively; P<0.001). Furthermore, significant correlations were observed between cholesterol and all ceramides except for C24:1 ceramide. Fasting insulin and HOMAIR correlated directly with ceramides C14:0, C18:0 and C24:1. For assessing HS, a cut-off points of 10.3 nmol/L for C14:0 Ceramide reported a sensitivity of 92.7% and a specificity of 73.5% when normal weight and obesity groups (n=52) were compared against obesity + HS group (n=42). Positive and negative predictive values were 77.5% and 90.2%, respectively.

Conclusions: plasma ceramides are closely associated with hepatic steatosis in adolescents. C14:0 ceramide could be a novel biomarker of HS independently of obesity.

Figure 1. Obesity and insulin resistance increase the production of pro-inflammatory cytokines and free fatty acids delivery to the liver that promotes ceramides production. In the hepatocyte ceramides increase apoptosis through ROS mechanism. The increase of ceramides in the liver facilitate its release into the circulation, which in turn affects insulin sensitivity in muscle tissue. IR= insulin resistance, AT= adipose tissue, FFA= free fatty acids, ROS= reactive oxygen species. 

Irina A Pikuleva studies cholesterol metabolism and cytochrome P450 enzymes whose role is to generate more polar forms of cholesterol for cellular elimination or regulation. Her laboratory focuses on the role of cholesterol-related proteins in retinal structure and function; and potential of CYP46A1 as a pharmacologic target for Alzheimer's disease. The ultimate goal of these studies is to identify new strategies for treatment of diseases of the eye (age-related macular degeneration and diabetic retinopathy) and the brain (Alzheimer’s disease). In search for these new strategies, the Pikuleva laboratory evaluates existing medications with a goal of drug re-purposing.

Statement of the Problem: Alzheimer’s disease (AD) is a progressive brain disorder and the most common cause of dementia among older adults. Currently, there is no cure for AD with all anti-AD treatments being only symptomatic. Cytochrome P450 46A1 (CYP46A1) converts cholesterol to 24-hydroxycholesterol and thereby initiates the major pathway for cholesterol elimination from the brain. CYP46A1 also controls the rate of cholesterol biosynthesis in the brain and the rate of cerebral cholesterol turnover. Studies of mouse models of AD showed that increasing CYP46A1 expression in the brain by genetic means ameliorates the manifestations of AD such as amyloid burden and cognitive deficits. We were able to enhance CYP46A1 activity pharmacologically in vitro and in mice by discovering that CYP46A1 has an allosteric site, binding to which of some non-cholesterol related drugs (e.g., the anti-HIV medication efavirenz) increases the rate of CYP46A1-mediated cholesterol 24-hydroxylation. The purpose of this study was to evaluate the effect of efavirenz, activating CYP46A1 at a very small dose, on 5XFAD mice, a model of rapid amyloidogenesis.

Methodology & Theoretical Orientation: Mice were treated with efavirenz for four and nine months and assessed for cholesterol homeostasis in the brain, amyloid pathology, cognition, and brain genome.

Findings: Efavirenz treatment: stably activated CYP46A1 and enhanced cholesterol turnover in the 5XFAD brain; reduced amyloid-β burden and microglia activation in the brain cortex and hippocampus; and affected mouse behavior in a taskand treatment time-specific manner with ultimately improving mouse performance in Morris water maze. The data obtained enabled generation of a mechanistic model, which unified EFV effects.

Conclusion & Significance: CYP46A1-mediated enhancement of cholesterol metabolism and turnover in the brain has a potential as a therapeutic strategy for AD and should be evaluated in a clinical trial.

Figure 1: Cholesterol metabolism in the brain.

Suzanne A Al-Bustan has completed her PhD in Human Genetics from the Duncan Guthrie Institute in Medical Genetics at Glasgow University in 1992. She is an Associate Professor of Human and Molecular Genetics in the Department of Biological Sciences and the Director of the joint MSc degree in Molecular Biology at Kuwait University. She has published numerous papers in reputed journals and has been active in both scientific research and supervision of several graduate students in the areas of genetics and molecular biology. Her main line of research expertise and emphasis is on genetic association of candidate genes in the lipid metabolism and transport with dyslipidemia in the Kuwaiti population.

Plasma lipids have been well documented to be influenced by lifestyle factors, such as diet, obesity, and physical activity, as well as genetic factors. Persistent fluctuation in levels of cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL), as well as decreased high-density-lipoprotein (HDL), often lead to clinical dyslipidemia which may consequently manifest into diabetes mellitus and/or coronary heart disease. Genetic association studies including genome wide association studies (GWAS) have attempted to elucidate the genetic and molecular mechanisms of dyslipidemia and have identified a limited number of candidate genes and pathways relevant to lipid metabolism and/or transport. However, conflicting results across different populations and ethnic groups have been reported. Variation in DNA sequences and candidate genes for blood lipid levels, therefore, remain unresolved. To demonstrate the importance of identifying cofounding gene variants in candidate gene loci relevant to specific ethnic groups and their role in dyslipidemia results from different studies conducted on the Kuwaiti population will be presented. Genetic association of the APOA promoter sequence, APOB and APOE common variants, and other related gene loci have been studied and positive association results from these will be presented. These include the association of the I -75G>A with increased levels of LDL-C and TC, APOB signal peptide and 3611 MspI polymorphisms with variation in TG levels and the APOE2 with LDL-C levels in the Kuwaiti population sampled. The nutrition of Kuwaitis is relatively high in fat and the lifestyle adapted by many in the Kuwaiti population often lead to an increase in many of the risk factors leading to heart disease especially dyslipidemia making this population ideal for genetic association studies. Important implications and conclusions drawn from these studies will be discussed.

Kawthar Alghamdi is pursuing her final year PhD in the School of Biomolecular and Biomedical Science, University College Dublin, Ireland under the supervision of Associate Professor Orina Belton. She received her MSc in Biotechnology from University College Dublin in 2013. Her previous work in the atherosclerosis research group at University College Dublin has shown that dietary administration of conjugated linoleic acid induces the regression of pre-established atherosclerosis via altering monocyte function. Her recent research has focused on the identification of distinct pathways regulated by conjugated linoleic acid that may yield further information as to how atherosclerosis can be reversed. To do this she has employed a comprehensive proteomic approach, to construct networks and identify key signaling pathways associated with altered monocyte/macrophage fate and function. Through this work she has identified and validated that TGFβ signaling mediates the atheroprotective effect of CLA.

The chronic recruitment of monocytes and their subsequent migration through the endothelium contribute to atherosclerotic plaque development, the underlying cause of heart attacks and stroke. A specific blend of conjugated linoleic acid (80:20 cis-9, trans-11: trans-10, cis-12-CLA) has the unique property of inducing regression of pre-established atherosclerosis in vivo via modulation of monocyte function. Currently, there are no therapeutic targets which induce regression of pre-established atherosclerosis. Therefore, understanding the mechanisms through which CLA 80:20 mediates its atheroprotective effect is important for more effective management of this disease. This study aimed to identify novel pathways regulated by CLA, which inhibits monocyte function using a proteomic approach. THP-1 monocytes were treated for 18 h with CLA blend, a lipid control Oleic acid (OA) or DMSO (n=3 per treatment group). Proteins were trypsin-digested prior to analysis by liquid chromatography coupled to high resolution, high mass accuracy Orbitrap mass spectrometry. Global proteomic protein identities and relative quantitation were determined in a label-free approach, using the MaxQuant, Perseus and IPA suite of programs. A total of more than 1500 proteins were identified by mass spectrometry across the experimental groups using Perseus. Following statistical analysis using the t-test, 121 proteins were found to be significantly altered following treatment with CLA 80:20 compared to the control (DMSO). 103 proteins were unique to CLA blend and not altered by OA. Subsequent bioinformatics analysis of the regulated proteins showed enrichment of the TGFβ signalling pathway. Validation of proteomic analysis was performed by Western blot analysis of THP-1 monocytes. Our data revealed that CLA 80:20 blend regulates the TGFβ signaling pathway in monocytes. This work contributes to our understanding of the atheroprotective pathways regulated by CLA 80:20 which impacts on monocyte/macrophage fate.

Figure 1: Global Proteomic Analysis: Significant proteins which were altered by CLA blend compared to control were determined by Student’s t-test, where considered significant a p-value < 0.05 (A) Hierarchical clustering global proteomic analysis exhibited only the 121 proteins which were statistically different with CLA relative to the control DMSO, 63 proteins down- regulated (green) and 85 proteins were upregulated(red), it was determined by Student’s t-test, where p< 0.05 flowing with z-score normalization of the median value of logarithmized intensities (Log 2) of each protein profile. Statistical analysis was performed in Perseus software (version 1.5.0.15). (B) Expression profile of the two hierarchical clusters, which were statistically different compare to the control group DMSO. (C) Common and unique number of proteins regulated by CLA blend and OA which were significantly changed compared to the control DMSO for both groups.