Apolipoprotein and lrp1-based peptides as new therapeutic tools in atherosclerosis

Altres ajuts: Fundació MARATÓ TV3 (201521-10)Altres ajuts: Fundación BBVAAltres ajuts: Sociedad Española de ArteriosclerosisApolipoprotein (Apo)-based mimetic peptides have been shown to reduce atherosclerosis. Most of the ApoC-II and ApoE mimetics exert anti-atherosclerotic effects by improving lipid profile. ApoC-II mimetics reverse hypertriglyceridemia and ApoE-based peptides such as Ac-hE18A-NH2 reduce cholesterol and triglyceride (TG) levels in humans. Conversely, other classes of ApoE and ApoA-I mimetic peptides and, more recently, ApoJ and LRP1-based peptides, exhibit several antiatherosclerotic actions in experimental models without influencing lipoprotein profile. These other mimetic peptides display at least one atheroprotective mechanism such as providing LDL stability against mechanical modification or conferring protection against the action of lipolytic enzymes inducing LDL aggregation in the arterial intima. Other anti-atherosclerotic effects exerted by these peptides also include protection against foam cell formation and inflammation, and induction of reverse cholesterol transport. Although the underlying mechanisms of action are still poorly described, the recent findings suggest that these mimetics could confer atheroprotection by favorably influencing lipoprotein function rather than lipoprotein levels. Despite the promising results obtained with peptide mimetics, the assessment of their stability, atheroprotective efficacy and tissue targeted delivery are issues currently under progress

High Density Lipoproteins: From Biological Understanding to Clinical Exploitation

Pharmacology/Toxicology; Molecular Medicine; Human Physiology; Immunology; Cell Biolog

Interfacial properties of the apolipoprotein Cs: implications for the regulation of lipoprotein catabolism and atherosclerosis

The risk of cardiovascular disease increases with elevated plasma levels of very-low density lipoproteins (VLDL) and chylomicrons. The human apolipoprotein Cs (apo C1, C2, C3) are small secretory proteins that circulate in plasma and play unique roles in the metabolism of VLDL and chylomicrons. ApoC2 is the required cofactor for lipoprotein lipase (LPL) which hydrolyzes plasma triacylglycerol. ApoC3 promotes VLDL synthesis in hepatocytes and both apoC1 and apoC3 inhibit LPL. The molecular details of these processes are largely unknown, but we hypothesized that apoC functions depend on protein structure, protein:lipid interactions, and surface pressure. Each apoC contains amphipathic N- and C-terminal helices that bind to and remodel lipid surfaces. Surface pressure—or the density of amphipathic molecules—increases significantly as LPL hydrolyzes triacylglycerol in VLDL. To probe the effects of protein structure and surface pressure on protein:lipid interactions, we used wild-type and point mutant variants of the apoCs, which differed in helical content and hydrophobicity. We used Oil-Drop Tensiometry to characterize the adsorption, conformational rearrangement, and desorption of each protein at lipid/water interfaces that mimic the core and surface of VLDL. This technique measured the effect of protein adsorption on surface pressure, and the surface area and pressure response of protein/lipid/water interfaces to volume changes that mimic lipogenic and lipolytic processes. We showed that the degree of protein amphipathic α-helical structure correlated with lipid affinity and provide a model for phenotypes in subjects with point mutations in apoC2 and apoC3. Each apoC exhibited multiple, pressure-dependent conformations at lipid surfaces, which indicates that the C-terminus of apoC2 likely desorbs from lipid at higher pressures to interact with LPL. ApoC3 exhibited a marked preference for lipid in the VLDL core, which provides novel insight into its role in VLDL assembly and secretion

Effects of phenol-enriched virgin olive oils on HDL functionality in hypercholesterolemic subjects

Els nivells de lipoproteïnes de baixa densitat (HDL) estan inversament relacionats amb la malaltia cardiovascular. No obstant, estudis d’intervenció i d’associacions genètiques assenyalen que la funcionalitat o qualitat de l’HDL és un paràmetre de major importància que la quantitat d’HDL. S’han descrit diversos efectes ateroprotectors dels compostos fenòlics (CFs) de l‘oli d’oliva verge (OOV). Conseqüentment, la síntesi d’OOV funcionals (OOVFs) enriquits amb CFs podria ser una bona estratègia per incrementar el consum de CFs sense incrementar la ingesta calòrica. La hipòtesi del present treball és que el consum sostingut d’OOVFs enriquits amb els seus propis CFs o amb ells més CFs complementaris propis de la farigola podria modificar les propietats fisicoquímiques de l’HDL i la distribució de les subclasses d’HDL, incrementant així la funcionalitat de l’HDL en individus hipercolesterolèmics. Dues intervencions aleatoritzades, controlades, doble cec i creuades foren realitzades dins el context de l’estudi “Virgin Olive Oil and HDL Functionality”. En la intervenció d’ingesta aguda, els participants ingeriren una dosi d’OOVFs enriquits amb els seus propis CFs a diferents concentracions (250-750 ppm de CFs). En la intervenció d’ingesta sostinguda, individus hipercolesterolèmics ingeriren OOVFs enriquits amb CFs a diferents concentracions i de diferent origen (80-500 ppm de CFs de l’oli d’oliva o combinats amb CFs de la farigola) durant tres setmanes. Els resultats mostraren que aquests OOVFs milloren la distribució de les subclasses d’HDL i diversos ratis aterogènics, incrementen la presència d’antioxidants en HDL, exerceixen un efecte beneficiós en la família de les paraoxonases i incrementen l’eflux de colesterol, d’acord amb la quantitat i l’origen dels CFs ingerits. Conseqüentment, aquests OOVFs podrien considerar-se nutracèutics apropiats per a augmentar la funcionalitat de l’HDL i com a una eina complementaria per al tractament d’individus hipercolesterolèmics.Los niveles de lipoproteínas de alta densidad (HDL) están inversamente relacionados con la enfermedad cardiovascular. No obstante, estudios de intervención y de asociaciones genéticas señalan que la calidad funcional de la HDL es un parámetro de mayor importancia que la cantidad de HDL. Se han descrito diversos efectos ateroprotectores de los compuestos fenólicos (CFs) del aceite de oliva virgen (AOV). Por consiguiente, la síntesis de AOV funcionales (AOVFs) enriquecidos con CFs podría ser una buena estrategia para incrementar el consumo de CFs sin incrementar la ingesta calórica. La hipótesis del presente trabajo es que el consumo sostenido de AOVFs enriquecidos con sus propios CFs o con ellos más CFs complementarios propios del tomillo podría modificar las propiedades fisicoquímicas de la HDL y la distribución de las subclases de HDL, incrementando así la funcionalidad de la HDL en sujetos hipercolesterolémicos. Dos intervenciones aleatorizadas, controladas, doble ciego y cruzadas fueron realizadas en el contexto del estudio “Virgin Olive Oil and HDL Functionality”. En la intervención de ingesta aguda, los participantes ingirieron una dosis de AOVFs enriquecidos con sus propios CFs a diferentes concentraciones (250-750 ppm de CFs). En la intervención de ingesta sostenida, sujetos hipercolesterolémicos ingirieron AOVFs enriquecidos con CFs a diferentes concentraciones y de diferente origen (80-500 ppm de CFs del aceite de oliva o combinados con CFs del tomillo) durante tres semanas. Los resultados mostraron que estos AOVFs mejoran la distribución de las subclases de HDL y varios ratios aterogénicos, incrementan la presencia de antioxidantes en HDL, ejercen un efecto beneficioso en la familia de las paraoxonasas e incrementan el eflujo de colesterol, de acuerdo con la cantidad y el origen de los CFs ingeridos. Consecuentemente, estos AOVFs podrían considerarse nutracéuticos apropiados para acrecentar la funcionalidad de la HDL y como una herramienta complementaria para el tratamiento de sujetos hipercolesterolémicos.High-density lipoprotein (HDL) cholesterol levels are inversely related to cardiovascular disease development. However, data from gene association and intervention studies lead to consider the functional quality of HDL as a more important parameter than the quantity of HDL. Phenolic compounds (PCs) from virgin olive oil (VOO) have been reported to hold atheroprotective functions. Therefore, the tailoring of functional VOOs (FVOO) enriched with PCs has been postulated as an interesting strategy to increase the daily PCs intake without increasing the caloric intake. The aim of the present thesis is that the sustained intake of FVOOs enriched with its own PCs or with them plus complementary ones from thyme may modify the physicochemical properties of HDL particles, and may promote changes in HDL subclasses distribution leading to the consequent enhancement of HDL functionality in hypercholesterolemic subjects. Two randomized, controlled, double-blind, and crossover interventions were conducted within the frame of the “Virgin Olive Oil and HDL Functionality” study. In an acute-intake intervention, participants ingested a single dose of FVOOs enriched with their own PCs but differing in the phenolic content (250-750 ppm of PCs). In a sustained-intake intervention, hypercholesterolemic participants ingested VOOs differing in PCs source and content (80-500 ppm of PCs from olive oil or combined with those from thyme) for three weeks. The results revealed that these FVOOs improve HDL subclasses profile and their associated atherogenic ratios, increase antioxidants content in HDL, exert a beneficial impact on paraoxonases family, and increase cholesterol efflux, according to PCs content and source in the FVOOs tested. Therefore, the tailoring of FVOOs could be a good nutraceutical to enhance the functionality of HDL and a complementary tool for the management of hypercholesterolemic individuals

Regulation of the Pregnane X Receptor Signaling Pathway

Liver-enriched nuclear receptors (NRs) collectively function as metabolic and toxicological `sensors' that mediate liver-specific gene-activation in mammals. NR-mediated gene-environment interaction regulates important steps in the hepatic uptake, metabolism and excretion of glucose, fatty acids, lipoproteins, cholesterol, bile acids, and xenobiotics. While it is well-recognized that ligand-binding is the primary mechanism behind activation of NRs, recent research is revealing that multiple signal transduction pathways modulate NR-function in liver. The interface between specific signal transduction pathways and NRs helps to determine their overall responsiveness to various environmental and physiological stimuli. The pregnane x receptor (PXR, NR1I2) was identified in 1998 as a member of the NR superfamily of ligand-activated transcription factors. PXR is activated by a broad range of lipophilic compounds in a species-specific manner. The primary function ascribed to PXR is the homeostatic control of steroids, bile acids, and xenobiotics. This function is mediated through PXR's ability to coordinately activate gene expression and regulate the subsequent activity of phase I and phase II metabolic enzymes, as well as several membrane transporter proteins. While PXR likely evolved primarily to protect the liver from toxic assault, its activation also represents the molecular basis for an important class of drug-drug, herb-drug, and food-drug interactions. While ligand binding is the primary mode of PXR activation, several signal transduction pathways interface with the PXR protein to determine its overall responsiveness to environmental stimuli. Multiple signaling pathways modulate the activity of PXR, likely through direct alteration of the phosphorylation status of the receptor and its protein cofactors. Therefore, specific combinations of ligand binding and cell signaling pathways affect PXR-mediated gene activation and determine the overall biological response. This dissertation contributes to the molecular understanding of the regulation of PXR by novel agonists, cAMP-dependent protein kinase (PKA) signaling, and phosphorylation. The results presented here were primarily obtained from mouse and tissue culture systems. This dissertation identifies Tian Xian, a traditional Chinese herbal anti-cancer remedy, as a novel PXR activator. This evidence suggests that Tian Xian should be used cautiously by cancer patients taking chemotherapy due to its potential to increase the metabolism of co-administered medications. In addition, data presented here show that activation of PKA signaling modulates PXR activity in a species-specific manner. It is further revealed that PXR exists as phospho-protein in vivo and that the activation of PKA signaling modulates the phospho-threonine status of PXR. Finally, the potential phosphorylation sites within the PXR protein are identified. These phosphorylation sites are characterized, using a phosphomimetic and phospho-deficient site-directed mutagenesis based approach, based on their ability to modulate PXR activity. Taken together, the work presented in this dissertation contributes to understanding the interface between ligands, signal transduction pathways and PXR activity, which is critical for the development of safe and effective therapeutic strategies

Regulation of lipid metabolism in adipocytes and hepatocytes by hexarelin through scavenger receptor CD36

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides.Growth hormone releasing peptides (GHRPs) are small synthetic peptides aimed at stimulating GH release from the pituitary through their binding to ghrelin receptor known as growth hormone secretagogue receptor 1a (GHS-R1a). Using the GHRP, hexarelin to study tissue distribution of GHS-R1a and its GH-independent effect, it was observed that hexarelin was capable of binding to a second receptor identified as scavenger receptor CD36. While having multiple ligands, CD36 is mainly known for binding and internalizing oxLDL and long chain fatty acids. CD36 is thought to play a detrimental role in macrophage derived foam cell formation and development of atherosclerosis. Previously, we have shown that in macrophages, expressing both GHS-R1a and CD36, hexarelin promoted an activation of PPARƔ via GHS-R1a but also through its binding to CD36. This activation led to the induction of the LXRα-ABC transporters pathway and an increase in cholesterol efflux, reducing lipid-laden macrophage content. This positive effect on macrophages was reproduced in apolipoprotein E-null mice on a high fat diet treated with hexarelin. A significant reduction in the size of atherosclerotic lesions was observed while similar increases in the expression of PPARƔ, LXRα and ABC transporters occurred in isolated peritoneal macrophages. CD36 also plays a role in fatty acid uptake, and to further investigate the impact of the interaction of hexarelin with CD36, we aimed at evaluating the role of CD36 in regulating lipid metabolism in cells devoid of GHS-R1a such as adipocytes and hepatocytes. In the present thesis, we demonstrated through its interaction with hexarelin, the ability of CD36 to decrease intracellular lipid content in both adipocytes and hepatocytes. In adipocytes, hexarelin was able to increase the expression of several genes involved in fatty acid mobilization, fatty acid oxidation but also to induce the expression of the thermogenic markers, PGC-1α and UCP-1. In addition, hexarelin increased the expression of genes involved in mitochondrial biogenesis which was accompanied by mitochondrial morphological changes in agreement with what is usually seen in highly oxidative cells. In support of these findings, we also observed an increase in the activity of cytochrome c oxidase (a component of the respiratory chain) which could reflect an increase in oxidative phosphorylation. The results generated with cultured white adipocytes suggest the ability of hexarelin to promote changes toward a brown fat-like phenotype which also occurred in vivo and was dependent on the presence of CD36. In hepatocytes, CD36 was capable of regulating cholesterol metabolism by rapidly phosphorylating LKB1 and AMPK which subsequently resulted in the inactivating phosphorylation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Hexarelin via CD36 also induced the recruitment of insig-2 to HMGR, the committed step in HMGR degradation while lifting the exerted inhibitory effect of Erk on nuclear receptor PPARƔ activity, and promoting the recruitment of AMPK to PPARƔ coactivator PGC-1α, suggesting an enhanced transcriptional potential of PPARƔ. The results generated during my graduate studies represent unique and novel mechanisms by which CD36 is capable of regulating lipid metabolism

LRH-1 as a target for the development of new breast cancer therapies

Estrogen drives the growth and development of estrogen receptor alpha (ERα) positive breast cancer and ERα is the target for hormonal therapies that inhibit its activity. A substantial proportion of patients become resistant to these therapies, demonstrating a need for new therapies. Gene expression microarray studies have been performed with a view to identifying potential novel therapeutic targets, biomarkers or forming the basis of identifying a molecular signature for endocrine resistance. These studies have identified candidate genes whose expression is altered in models of endocrine resistance. Investigation of the molecular pathways particularly highlights cell survival and regulation of apoptosis and indicates that these pathways play a key role in the development of resistance. Microarray analysis also identified the liver receptor homolog 1 (LRH-1, NR5A2), a member of the nuclear receptor superfamily of transcription factors, as an estrogen regulated gene in MCF7 cells. Functional analysis showed that LRH-1 regulates breast cancer cell growth, acting in part by regulating ERα expression. Gene expression profiling of MCF-7 cells following RNAi for LRH-1 identified LRH-1 regulated genes. LRH-1 is known to regulate expression of CYP19A1 (aromatase), responsible for estrogen biosynthesis through the aromatisation of aromatase. Together, our findings identify LRH-1 as a potential therapeutic target for breast cancer treatment. Results of screening for small molecule inhibitors of LRH-1 will be presented, together with analysis of gene expression profiling for LRH-1 regulated genes.Open Acces

PPARs as Key Mediators of Metabolic and Inflammatory Regulation

Mounting evidence suggests a bidirectional relationship between metabolism and inflammation. Molecular crosstalk between these processes occurs at different levels with the participation of nuclear receptors, including peroxisome proliferator-activated receptors (PPARs). There are three PPAR isotypes, α, β/δ, and γ, which modulate metabolic and inflammatory pathways, making them key for the control of cellular, organ, and systemic processes. PPAR activity is governed by fatty acids and fatty acid derivatives, and by drugs used in clinics (glitazones and fibrates). The study of PPAR action, also modulated by post-translational modifications, has enabled extraordinary advances in the understanding of the multifaceted roles of these receptors in metabolism, energy homeostasis, and inflammation both in health and disease. This Special Issue of IJMS includes a broad range of basic and translational article, both original research and reviews, focused on the latest developments in the regulation of metabolic and/or inflammatory processes by PPARs in all organs and the microbiomes of different vertebrate species

Drug Discovery

Natural products are a constant source of potentially active compounds for the treatment of various disorders. The Middle East and tropical regions are believed to have the richest supplies of natural products in the world. Plant derived secondary metabolites have been used by humans to treat acute infections, health disorders and chronic illness for tens of thousands of years. Only during the last 100 years have natural products been largely replaced by synthetic drugs. Estimates of 200 000 natural products in plant species have been revised upward as mass spectrometry techniques have developed. For developing countries the identification and use of endogenous medicinal plants as cures against cancers has become attractive. Books on drug discovery will play vital role in the new era of disease treatment using natural products

Phytochemical Omics in Medicinal Plants

Medicinal plants are used to treat diseases and provide health benefits, and their applications are increasing around the world. A huge array of phytochemicals have been identified from medicinal plants, belonging to carotenoids, flavonoids, lignans, and phenolic acids, and so on, with a wide range of biological activities. In order to explore our knowledge of phytochemicals with the assistance of modern molecular tools and high-throughput technologies, this book collects recent innovative original research and review articles on subtopics of mechanistic insights into bioactivities, treatment of diseases, profiling, extraction and identification, and biotechnology