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E-ISSN 2013-410X | OPEN ACCESS JOURNAL | www.cat-science.cat

O P E N A ACCE S S VOLUME 13 | ISSUE 1 | JUNE 2017

Institut d’Estudis Catalans Barcelona · Catalonia


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Founded in 1999 by Salvador Reguant Science and Technology Section, IEC Address

CONTRIBUTIONS TO SCIENCE Institut d’Estudis Catalans Carme, 47 08001 Barcelona, Catalonia Tel. +34 932701620 Fax +34 932701180 Email: contributions@iec.cat Publisher Institut d’Estudis Catalans, Barcelona, Catalonia www.iec.cat e-ISSN (electronic): 2013-410X D.L.: B 36385-1999 © 2017 Institute for Catalan Studies and authors. Printed in Catalonia.

GENERAL INFORMATION CONTRIBUTIONS TO SCIENCE is the international journal of the Biological Sciences Section and the Science and Technology Section of the Institute for Catalan Studies (IEC). It is a peer-reviewed scientific journal, published twice a year by the IEC. It publishes reviews on topics of the highest interest in all branches of sciences. Transversal articles between sciences and humanities are also welcome. It was first published in 1999. Aims and scope

CONTRIBUTIONS TO SCIENCE is an Open Access journal that aims to promote the international dissemination of scientific research performed in Catalonia in any of its branches, both pure and applied. CONTRIBUTIONS TO SCIENCE publishes research performed in countries with linguistic, cultural and historic links with Catalonia. It also publishes scientific articles of international standing related to all such territories, especially considered as a whole. The journal also covers studies performed in all parts of the world by scientists from such countries and articles based on lectures imparted by invited foreign scientists. Preference will be given to original articles in the form of critical reviews that deal with the state of the art of a scientific field of current interest, by one or several authors. Such articles should summarize the development, the present situation and, where possible, future perspectives of a research area in which the author or authors have participated directly. The journal will also publish articles, short communications, notes and news items of international interest on historical, economic, social or political aspects of research in Catalonia and its areas of influence. Copyright and responsibilities

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THE ACADEMY OF CATALAN LANGUAGE AND THE INSTITUTE OF SCIENCES AND HUMANITIES The Academy of Catalan Language, founded in 1907, is the top academic corporation of the territories of Catalan language and culture, and has been a full member of the International Academic Union since 1922. The IEC has 186 full or emeritus members from throughout the linguistic territory, and 72 corresponding members that represent our institution’s relations with the international scientific community, and has 28 filial societies of all fields of knowledge, with a total membership of around 10.000 across the whole territory. In addition, 111 local research centres also belong to it, and this shows how well grounded the research community is, throughout our cultural territory. The IEC is the central institution in the Catalan cultural world. It was set up in 1907 at the initiative of the Diputació de Barcelona to “establish here scientific study centres specialising and working not just in education, but in producing science and aiding research.” In the following years, the Institute set up its various science departments. The Philology Department, directed by Pompeu Fabra, played a key role in establishing the rules of the Catalan language.


O P EVOLUME N 13A| ISSUE A1 |CJUNEC2017E S S

Institut d’Estudis Catalans Barcelona · Catalonia


OVolume P E 13 N |AIssue A 1C|CJune E 2017 SS

Editorial Board EDITOR-IN-CHIEF

Ricard Guerrero Biological Sciences Section, IEC

ASSOCIATE EDITOR

ASSOCIATE EDITOR

Salvador Alegret

Ramon Gomis

Science and Technology Section, IEC

Biological Sciences Section, IEC

PUBLICATIONS BOARD

Pere Puigdomènech Mercè Durfort Jaume Terrades Xavier Llimona Abel Mariné

This issue of Contributions to Science was supervised by the Biological Sciences Section of the Institute for ­Catalan Studies.

GOVERNING BOARD

Francesc González Sastre President

Abel Mariné VicePresident

Xavier Llimona Secretary

Joaquim Gosálbez Treasurer

EDITORIAL BOARD The Science and Technology and Biological Sciences Sections: Joaquim Agulló, Technical University of Catalonia • Josep Amat, Technical University of Catalonia • Francesc Asensi, University of Valencia • Damià Barceló, Spanish National Research Council (Barcelona) • Carles Bas, Institute of Marine Sciences-CSIC (Barcelona) • Pilar Bayer, University of Barcelona • Xavier Bellés, Spanish National Research Council (Barcelona) • Jaume Bertranpetit, Pompeu Fabra University (Barcelona) • Eduard Bonet, ESADE (Barcelona) • Joaquim Casal, Technical University of Catalonia • Alicia Casals, Technical University of Catalonia • Josep Castells, University of Barcelona • Jacint Corbella, University of Barcelona • Jordi Corominas, Technical University of Catalonia • Michel Delseny, University of Perpinya • Josep M. Domènech, Autonomous University of Barcelona • Mercè Durfort, University of Barcelona • Marta Estrada, Institute of Marine Sciences-CSIC (Barcelona) • Gabriel Ferraté, Technical University of Catalonia • Ramon Folch, Institute for Catalan Studies • Màrius Foz, Autonomous University of Barcelona • Jesús A. Garcia-Sevilla, University of the Balearic Islands • Lluís Garcia-Sevilla, Autonomous University of Barcelona • Joan Genescà, National Autonomous University of Mexico • Evarist Giné, University of Connecticut (USA) • Joan Girbau, Autonomous University of Barcelona • Pilar González-Duarte, Autonomous University of Barcelona • Francesc González-Sastre, Autonomous University of Barcelona • Joaquim Gosálbez, University of Barcelona • Albert Gras, University of Alacant • Gonzalo Halffter, National Polytechnic Institute (Mexico) • Lluís Jofre, Technical University of Catalonia • Joan Jofre, University of Barcelona • David Jou, Autonomous University of Barcelona • Ramon Lapiedra, University of Valencia • Angel Llàcer, Hospital Clinic of Valencia • Josep Enric Llebot, Autonomous University of Barcelona • Jordi Lleonart, Spanish National Research Council (Barcelona) • Xavier Llimona, University of Barcelona • Antoni Lloret, Institute for Catalan Studies • Abel Mariné, University of Barcelona • Joan Massagué, Memorial Sloan-Kettering Cancer Center, New York (USA) • Federico Mayor-Zaragoza, Foundation for a Culture of Peace (Madrid) • Adélio Machado, University of Porto (Portugal) • Gabriel Navarro, University of Valencia • Jaume Pagès, Technical University of Catalonia • Ramon Parés, University of Barcelona • Angel Pellicer, New York University (USA) • Juli Peretó, University of Valencia • F. Xavier Pi-Sunyer, Harvard University (USA) • Norberto Piccinini, Politecnico di Torino (Italy) • Jaume Porta, University of Lleida • Pere Puigdomènech, Spanish National Research Council (Barcelona) • Jorge-Óscar Rabassa, National University of La Plata (Argentina) • Pere Roca, University of Barcelona • Joan Rodés, University of Barcelona • Joandomènec Ros, University of Barcelona • Claude Roux, University of Aix-Marseille III (France) • Pere Santanach, University of Barcelona • Francesc Serra, Autonomous University of Barcelona • David Serrat, University of Barcelona • Boris P. Sobolev, Russian Academy of Sciences, Moscow, Russia • Carles Solà, Autonomous University of Barcelona • Joan Antoni Solans, Technical University of Catalonia • Rolf Tarrach, University of Luxembourg • Jaume Terradas, Autonomous University of Barcelona • Antoni Torre, Obra Cultural de l’Alguer • Josep Vaquer, University of Barcelona • Josep Vigo, University of Barcelona • Miguel Vilardell, Autonomous University of Barcelona • Jordi Vives, Hospital Clinic of Barcelona


OVolume P E 13 N |AIssue A 1C|CJune E 2017 SS

Contents

Gonzàlez Sastre, F.

7

Introduction

Arús, P.

9

Molecular markers for plant genetics and breeding

Canyelles, M.; Cedó, L., Julve, J.; 17 Escolà-Gil J.C.; Blanco-Vaca, F.

HDL function and novel HDL-targeted therapies for preventing atherosclerotic cardiovascular disease: From mouse models to human disease

Capellá, G.

33 Bellvitge Biomedical Research Institute: research centre for cancer, neurosciences and translational medicine

Llovet, J.M.

37

Translational medicine in Catalonia: the case of liver oncology


CONTRIBUTIONS to SCIENCE 13(1):7 (2017) Institut d’Estudis Catalans, Barcelona, Catalonia www.cat-science.cat

Introducció

Introduction

La publicació d’aquest número amb la seva estructura i els seus continguts respon als objectius senyalats en reunions passades de la nostra institució sobre Contributions to Science: les modificacions del format, els continguts i la seva presentació. En efecte, cal donar a conèixer els centres de recerca del nostre país i molt especialment els nostres investigadors i la seva apreciable tasca continuada. Si això fou important el 1999 quan es creà Contributions to Science, actualment, amb l’esplèndid desenvolupament dels darrers anys, constitueix una prioritat per l’IEC com a centre referent dels coneixements científics dels Països Catalans. En moments de canvi i renovació cal fer memòria dels orígens i dels seus protagonistes. Contributions to Science, com a publicació científica periòdica, va néixer de la idea d’un dels membres de la Secció de Ciències i Tecnologia, en Salvador Reguant i Serra, que creia en la necessitat d’una publicació de les Seccions de Ciències de l’IEC que tingués l’ambició i la capacitat d’exposar amb eficàcia i internacionalment en el món de la ciència l’activitat dels Països Catalans. El 1999 el seu projecte es feu realitat amb la publicació del primer número de Contributions to Science. Aquell primer número conté una introducció-presentació de l’aleshores president de l’IEC, Manuel Castellet, que subratllà l’important desenvolupament quantitatiu i qualitatiu de la recerca en la nostra àrea cultural i indicà que en aquells àmbits que prèviament existia una tradició de recerca, els equips obtingueren un gran renom, però també que havien aparegut qualificats investigadors on prèviament hi havia hagut poca activitat. L’objectiu de la nova publicació, indicat pel president de l’IEC, fou donar difusió internacional de la recerca realitzada en els països de llengua catalana en totes les branques de la ciència, pura o aplicada En aquests darrers anys, quan la recerca científica en els nostres països ha crescut considerablement en volum, qualitat i en reconeixement internacional, el progrés també general de la microelectrònica ha facilitat la connectivitat i la digitalització amb un gran impacte en la comunicació general però molt especialment en la comunicació científica. En aquest sentit l’ús del paper s’ha anat reduint en favor de la comunicació immediata en l’àmbit digital. L’ambició de la renovació de Contributions to Science és continuar els objectius generals de donar a conèixer la recerca dels nostres àmbits amb una nova política de continguts i formes de comunicació i que continuï sent la publicació de les Seccions de Ciències de l’IEC. En un període de canvis cal agrair la constància a tots aquells que feren possible l’aparició de la publicació i molt especialment la seva continuïtat. L’índex del número actual, que pretén encetar un patró a seguir, conté la presentació d’un centre de recerca repetidament acreditat pels resultats obtinguts i tres treballs de revisió dels darrers anys de grups de recerca que han merescut l’atenció del món de la recerca local i internacional.

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The publication of the present with its structure and its contents responds to the objectives indicated in previous meetings of our institution on Contribution to Science: the modifications of the format, the content and its presentation. In fact, it is necessary to inform about the research centers of our country, and especially our scientists and their appreciable continued work. If that was important in 1999 when Contributions to Science was created, nowadays, with the splendid development of recent years, it is a priority for IEC as a reference institution for the scientific know­ledge of Catalan Countries. In times of change and renovation, we must remember the origins and the promoters. Contributions to Science, a periodical scientific publication of the Sections of Science of the IEC was born from an idea of one of the members of the Section of Sciences and Technology, Salvador Reguant i Serra that believed in the need for a publication of the Sections of Sciences of the IEC with the ambition and the capacity to expose the activity of the Catalan countries with efficiency and internationally to the world of science. In 1999 his project came true with the publication of the first number of Contributions to Science. That first issue contained an introduction-presentation by the then President of the IEC, Manuel Castellet, who underlined the important quantitative and qualitative development of research in our cultural area. He indicated that in those areas that previously there was a tradition of research, the teams had a great reputation, but also that they had appeared qualified researchers where previously there had been little activity. The objective of the new publication, indicated by the President of the IEC, was to disseminate international research into Catalan-speaking countries in all branches of science, pure or applied. In recent years, when scientific research in our countries has grown considerably in volume and quality, and in international recognition, the general progress of telecommunications has facilitated connectivity and digitization with a great impact on general communication but very much especially in scientific communication. In this sense, the use of paper has been reduced in favor of immediate communication in the digital environment. The ambition of the renewal of Contribution to Science is to continue the general objectives of presenting the research of our fields with a new policy of contents and forms of communication of the publications of the Sections of Sciences of the IEC. In a period of changes, the continuity of the science publication of the IEC must be celebrated, especially thanks to all those who make possible the creation of the publication and especially its continuity. The index of the current number that aims to be a model to continue, contains the presentation of a research center repeatedly accredited by the results obtained with the general objective of going to the different research centers. Also three works of review of the last years of research groups that have earned the attention of the world of local and international research. Francesc González Sastre, President of the Section of Life Sciences (until November 2017) of IEC

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RESEARCH REVIEWS Institut d’Estudis Catalans, Barcelona, Catalonia

O P E N A ACCE S S

www.cat-science.cat

CONTRIB SCI 13(1):9-15 (2017) doi:10.2436/20.7010.01.256

Molecular markers for plant genetics and breeding Pere Arús IRTA, Centre de Recerca en Agrigenòmica CSIC-IRTA-UAB-UB. Campus UAB, Edifici CRAG; Cerdanyola del Vallès (Bellaterra); 08193 Barcelona Abstract. The development of molecular markers has been a major step forward in understanding the genetic basis of phenotypic diversity, and in measuring the amount and organization of genotypic polymorphisms in wild and cultivated plant species. The progress in molecular technologies of the last 50 years has lead from a few isozyme markers to a virtually unlimited number of DNA-based markers that are highly polymorphic, codominant, ubiquitous in the genome, and can be obtained by relatively cheap and simple technologies. With markers it has been possible to introduce linkage as a new dimension in genetic analysis, allowing map construction, dissection of quantitative traits, association genetics, positional cloning of genes of interest and the study of genome evolution based on the comparison of the genome positions of homologous markers. These developments have led to multiple applications for plant breeding, including cultivar fingerprinting, major gene or QTL (quantitative trait loci) assisted selection, whole-genome selection in backcross programs, and genomic selection, which have been adopted by most breeders as tools to enhance the efficiency of conventional methods of plant improvement. This paper summarizes the research conducted in Catalonia that has been pioneering in this area at the international level, particularly for horticultural crops.

Resum. Els marcadors moleculars han permès un dels majors avenços recents en la comprensió de la base genètica de la diversitat fenotípica i la mesura de la variació genètica en plantes silvestres i cultivades. En els darrers 50 anys hem passat d’unes desenes d’isoenzims a un nombre virtualment il·limitat de marcadors d’ADN altament polimòrfics, codominants, presents a tot arreu del genoma i que poden ser obtinguts amb tecnologies senzilles i barates. Els marcadors han introduït el lligament com una nova dimensió en l’anàlisi genètica, permetent la construcció de mapes, la dissecció de caràcters quantitatius, la genètica d’associació, el clonatge posicional de gens d’interès i l’estudi de l’evolució dels genomes basada en la comparació de mapes. Els marcadors han estat també útils per la millora genètica, permetent la identificació molecular de genotips, la selecció assistida amb marcadors de gens majors i QTLs (loci de caràcters quantitatius), la selecció de tot el genoma en programes de retroencreuament i la selecció genòmica, que han estat adoptats com eines que augmenten l’eficiència dels mètodes convencionals. Aquest article resumeix la recerca realitzada a Catalunya en aquest àmbit que ha estat pionera a nivell internacional, en particular als fruiters i hortalisses.

Keywords: molecular markers, genetic variability, marker-assisted selection, plant improvement Correspondence:

Pere Arús pere.arus@irta.cat

The evolution of molecular markers: from isozymes to next generation sequencing-based markers After the rediscovery of Mendel’s laws at the beginning of the 20th century, models of genetics, evolution and pop-

e-ISSN: 2013-410X

ulation genetics drawn up by pioneers such as R. Fisher, J.B.S. Haldane and S. Wright were essentially theoretical and their experimental validation had two major drawbacks: the observed variability was generally determined by dominant genes, and including linkage in the models was difficult. Only two phenotypes can be observed for a

CONTRIBUTIONS to SCIENCE 13(1):9-15 (2017)


Molecular markers for plant genetics and breeding

single dominant A/a gene: the recessive class aa, and the dominant class A- (including the indistinguishable AA and Aa classes). This precludes a proper analysis of the adjustment of its segregation to a Hardy-Weinberg equilibrium or its departures caused by mutation, selection, migration, inbreeding or random drift. With regard to the second drawback, linkage, not intuited by Mendel, was discovered relatively early in the 20th century (T.H. Morgan and A.H. Sturtevant) and integrated in population genetics models although essentially as two or three-locus interactions. Linkage analysis was difficult because of the dominance of the genes available, and the fact that they were often mutants, natural or induced, where the aa genotype frequently had a deleterious effect on the phenotype. Working with many of these genes – also called major genes or morphological markers - was extremely laborious as they rarely segregated in natural populations, often requiring the complex and time consuming elaboration of plant stocks adequate to study their cosegregation and linkage. In short, the construction of linkage maps was an endeavor that required so much investment and time that it was restricted in plants to a few model crops: wheat, pea, maize, barley, rice and tomato. The first markers that represented a real change were isozymes, variants of specific enzymes that retained their activity but had a slightly different amino acid composition that modified their shape or electric charge. They could be separated by electrophoresis and their position identified as bands with histochemical stains. While they were discovered at the end of the 1950s, their use in plant genetics became significant 15 years after, increasing until the 1980s, when they began to be substituted by DNA markers. Enzymes with one or a few copies in the genome were ideal, as they produced few bands of easy genetic interpretation, each corresponding to a different allele (allozyme) of one enzyme-coding gene (isozyme). Their advantage was that it was possible to access a set of variable, codominant genes that did not usually interact with each other or with the environment, and that allowed the variability between different populations and species to be compared. Their main disadvantage was their low number: a few tens per species. In spite of this major limitation, isozymes opened up a new era in plant genetics and breeding. Using them made it possible to propose new goals in essentially two distinct domains: the analysis of genetic variability, and the use of markers in connection with linkage, i.e. to monitor changes occurring in neighboring genes - or by extension at the

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whole genome level - and to study evolution of the entire genome. Most of the new uses of markers for plant genetics and breeding were conceived and developed within a single decade (Tanksley and Orton, 1982), because the theoretical problems were well known and only required an adequate tool for their study. The genotypes of sets of isozymes in wild or cultivated populations provided extremely useful information on their level of variability and their history, and predicted their evolution under external constraints, including the evolution of breeding populations under selection. They also permitted a first comparison of the levels of variability of different species, established the importance of the mating system in the amount and distribution of variability across populations (Gottlieb 1981), and provided an elegant approach to identify species arising from recent polyploidy events (Gottlieb 1982). Many applications of isozymes emerged: the identification of cultivars or genotypes, important for protection of breeder’s rights or as quality control of propagating materials; tests for F1 seed purity, and evaluation of the parentage of individuals of unknown pedigree (Tanksley and Orton 1982). Other applications involved the use of isozymes linked to major genes of interest (marker-assisted selection) for earlier or more efficient selection, and the use of markers for rapid recovery of the recurrent genome in backcross programs (Tanksley et al. 1981). The dissection of quantitative characters in their component QTLs (quantitative trait loci) was first proposed at this time (Tanksley et al. 1982). Isozyme linkage groups in different species also suggested the conservation of genomic regions in broad evolutionary transects, which was the beginnings of comparative mapping (Tanksley and Orton 1982). The first marker type allowing a direct analysis of DNA variation was RFLP (restriction fragment length polymorphism) (Botstein et al. 1980). Codominant markers of high quality could be produced, with the advantages over isozymes in that their number was virtually unlimited, and RFLPs identified variation in nearly every genome region, coding or noncoding. RFLPs are produced by digesting the total DNA of an individual with a restriction enzyme, separating the resulting fragments by electrophoresis, and detecting the positions of specific fragments using a short radioactively-labeled DNA probe. The RFLP method is time-consuming and expensive, but allowed the construction of the first complete (saturated) maps of many species, with tomato (Bernatzky et al. 1986) being the first in a long series of maps. For the first time it was possible to use genetic strategies requiring whole genome coverage,

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$UºV

genomes of most of the important crop species are now sequenced or will be in the near future. Whole genome sequences mean a simplification of genetic analysis as they provide a template with which low coverage random sequences (resequences) of one or a set of genotypes can be aligned, allowing the detection of often millions of markers (SNPs and indels). Having large sets of markers facilitates the saturation of specific map regions and the fine mapping of target genes and QTLs. The whole set of genes of a species are located in the genome, making it very simple to associate their positions with that of specific major genes or QTLs already described. Sets of markers with coverage of the whole genome can be designed for any application to breeding: fingerprinting, linkage map construction, association analysis and genomic selection, among other uses. With resequencing, specific genome regions containing genes of interest can be explored, and their genetic polymorphism analyzed in individuals with different phenotypes, allowing strong genotype-phenotype inferences.

allowing full realization of what could only be sketched with isozymes, such as QTL analysis (Paterson et al. 1989) and comparative mapping (Ahn and Tanksley, 1993). The abundance of markers also made it possible to find them where needed, particularly in the neighborhood of genes of interest, resulting in a generalized use of marker-assisted selection (MAS), and for whole-genome selection to accelerate backcross breeding programs (Tanksley et al. 1989). New avenues were also opened, with the possibility of cloning genes by successive identification of markers that were close to their position, with the help of large-fragment DNA yeast and bacterial artificial chromosome libraries (YACs and BACs). The decade of the 1990s witnessed the development of many other kinds of markers that progressively substituted RFLPs, maintaining the high quality and number, with improvements in method simplicity and costs. Most were based on the polymerase chain reaction (PCR) along with sequencing technologies, especially Sanger sequencing using capillary electrophoresis, with markers more targeted to specific sequences or genome regions. Currently there are two main sources of markers based on the two major sources of DNA variability: SNPs (single-nucleotide polymorphisms) that detect single nucleotide substitutions, and SSRs (simple-sequence repeats), that use insertion/deletion polymorphisms in the widespread microsatellite sequences. New genetic analysis strategies were developed when the cost of genotyping large numbers of markers became reasonable. Bulked segregant analysis, a powerful approach to find markers close to specific genes of interest, was developed early on (Michelmore et al. 1991), and has been useful for finding markers for MAS or as a first step towards map-based cloning. Another key strategy was genome-wide association analysis, offering a wider range of variability for inheritance studies and allowing more precise mapping of genes/QTLs than with conventional biparental populations. Genotype-phenotype relationships can be predicted using a genotypically well characterized subset of individuals (the training population) from where a model of phenotypic prediction is tested and applied to a broader sample of individuals. This is the basis of genomic selection that has proved valuable in animal breeding, and promises to also be useful in plant species, particularly those with long-generation times such as fruit and forest trees. The first genome sequenced in plants was that of Arabidopsis in 2000, but with the advent of next generation sequencing devices, this has become relatively affordable and

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Marker analysis research in Catalonia Research at IRTA on horticultural crops This began in 1982 at the Cabrils Center of SIA (Servei d’Investigació Agraria) with the research facilities transferred to the Government of Catalonia from the INIA (Instituto Nacional de Investigaciones Agrarias). In 1985, IRTA (Institut de Recerca i Tecnologia Agroalimentàries) was created, that included the SIA and other agricultural research groups in Catalonia. The initial research objectives were to establish a laboratory adequate for the work with isozymes, and to use these markers to understand genetic variability and its applications in breeding programs at IRTA or other public or private parties. Recently (2011), researchers in the fields of genetics and genomics moved to CRAG (Centre de Recerca en Agrigenòmica), a consortium of IRTA, CSIC (the Spanish Consejo Superior de Investigaciones Científicas) and the two main Universities of Barcelona (UB and UAB) at the UAB Campus in Bellaterra. In the initial stages, peach, almond, hazelnut, walnut, carnation and carob were the target species, and we studied isozyme variability, inheritance and linkage. These data were useful for cultivar fingerprinting, of interest for quality control by nurserymen, and for identity testing for official organizations responsible for granting breeder’s rights. In collaboration with a seed company, we studied the use of isozymes for seed purity testing in a set of vegetable crops (tomato, pepper, melon, cucumber and eggplant). In all,

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Molecular markers for plant genetics and breeding

Korean cultivar selected to be one of the most genetically distant cultivated melon accessions based on marker data. We developed collections of the new SSR markers for our species of interest, mainly peach and other stone fruit, melon and strawberry, the latter a species with a complex octoploid genome for which we had a contract with a breeding company. SSRs were used to further saturate our reference maps, at the same time providing adequate markers to other research groups for the construction of maps in different populations and the study of other characters. Using maps from Prunus crops we found that all shared essentially the same genome and we were able to identify the positions of many major genes mapped in different populations from these species in a unique reference map (Dirlewanger et al. 2004). A method for mapping any marker using a reduced sample of plants (8) from this map was developed (Howad et al. 2005) and used in many other crops. In melon, a gene responsible for resistance to melon necrotic spot virus was map-based cloned (Nieto et al. 2006). Markers closely linked to disease resistances or other traits were developed for use in plant breeding, in the vegetable and fruit species in which we worked, some of which developed ourselves in collaboration with our industrial partners, and others developed by third parties that we adapted to our needs and conditions. Most of these markers have been adopted by breeders to aid faster and more efficient selection. SSRs were also useful to study the variability of crops and for fingerprinting due to their high polymorphism, and because they were mapped and could be selected to cover the whole genome at regular intervals. SSRs provided an insight to the evolution of peach after its domestication, at least five millennia ago in China. Genotypes of large collections of cultivars showed that the original Chinese landraces (Li et al. 2015) were much more variable than the occidental and oriental breeding materials due to the extent of erosion in the gene pool generated by inbreeding, selection and random drift. In the case of occidental cultivars, markers were also able to separate materials from the modern breeding programs and a group of traditional Spanish peach non-melting flesh genotypes: the latter were essentially homozygous due to the long periods when they were seed reproduced, being clonally reproduced by grafting only in their most recent history. An important outcome of these variability analyses was that they revealed the enormous power of SSRs for genotype fingerprinting. In 1996 a Spanish peach breeding company proposed we develop marker tests for charac-

isozymes were reliable and useful, but their use was highly dependent on the level of genetic variability of the species, given the limited number of marker loci available. For species with low variability, such as tomato, pepper and peach, the extent of the application was narrow, as the ability of the markers to discriminate was also low. The situation changed with the development of PCRbased markers, starting from 1990. The first of these, known as RAPDs (random amplified polymorphic DNA), had various quality problems (dominance and low repeatability), but they were so abundant that it was often possible to find a sufficiently reproducible polymorphism for certain simple applications. This was a solution for the F1 seed purity test analysis, where one or two polymorphic markers between the two inbred lines of a hybrid were sufficient for establishing a robust and efficient test. These results were rapidly adopted by a collaborating seed company: the DNA test reliably confirmed the quality of F1 hybrid seed lots from different origins within a short period of time. The alternative was a time consuming and error-prone grow-out test, which involved the phenotypic identification of sibs (usually selfing progeny of the female inbred) from a sample of plants of each lot grown in the greenhouse. Since then, most breeding companies have used DNA markers for seed purity tests, a major quality procedure. At that moment, RFLPs were the appropriate markers for constructing linkage maps, and we used these and isozymes to produce a saturated map for almond (Viruel et al. 1995), which was the first to be published and the most complete in stone fruit (Prunus). Between 1992 and 1996 we were granted a European project to construct maps for peach, cherry, almond and plum that I coordinated and which included French, Italian, British and Spanish partners. The approach was to elaborate a densely populated, high quality map for peach, and then construct maps of lower density in several populations of the other species using selected markers from the peach map. Given the low genetic variability of peach, an interspecific F2 almond x peach was chosen as the mapping population, which insured sufficient population and map coverage. This map (Joobeur et al. 1998) was adopted by the Prunus scientific community as the reference for the genus and was used to establish the chromosome terminology and orientation. There was similar development in melon, in which we strongly invested as it was high priority for one of our industrial partners. The first melon map was developed with RFLPs (Oliver et al. 2001), in the F2 from the cross between a line of the major cultivar produced in Spain (Pinyonet Piel de Sapo) and a

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for a given character), and being a first step towards mapbased cloning. The first NIL population we constructed was using the parents of the melon map, with ‘Piel de Sapo’ as the recurrent genotype (Eduardo et al. 2005). This population has been extensively used for the analysis of a diverse set of characters, including those related with fruit shape and weight, a quantitative resistance to cucumber mosaic virus, and to unravel the complexity of the climacteric vs. non climacteric ripening process in melon. Another NIL collection has been developed for diploid strawberry (Fragaria vesca), using the cultivar ‘Reine des Vallées’ as recurrent parent and an accession of F. bucharica as exotic donor (Urrutia et al. 2015a). With this collection it has been possible to find many major genes involved in different morphological and reproductive traits, and it is especially useful for the analysis of metabolites involved in fruit taste and aroma (Urrutia et al. 2015b). Breeding in perennial species, such as fruit or forest trees, is limited by their long intergeneration periods, such that conventional breeding schemes are slow and inefficient. In peach this is a limiting factor, as the species is highly monomorphic, so hinders the enrichment of its genome with new genes from exotic materials or wild relatives. In fact, none have been introduced in a commercial cultivar while genes for disease resistance or longer postharvest life are in great demand. We proposed a new marker-based breeding scheme, marker-assisted introgression (MAI), to transfer one specific chromosome fragment from an exotic donor into an elite peach germplasm in only two backcross generations. In this method, markers are used to select individuals with a few introgressions in a large backcross one (BC1) progeny, and those with only one introgression in BC2 detected. A small collection of BC1 lines with a few introgressions allows a first survey of the new variability provided by the donor parent, with the detection of some of the major genes or QTLs that are dominant for the donor parent allele or additive. We have shown that the process works correctly in the offspring of almond × peach crosses in only eight years (Serra et al. 2016). This has been also useful to generate a collection of NILs of almond fragments in the peach background that we expect to share with the scientific community in the next few years. We had an active role in sequencing full genomes for the species in which we are primarily interested, being involved in the creation of the sequencing consortia and participating directly in generation of the sequence, as was the case for strawberry (Shulaev et al. 2011) and peach (Verde et al. 2013). Given that three of the major genomes of the

terizing their varieties to use in the protection of breeder’s rights. Later, with GESLIVE (Gestión de Licencias Vegetales) - a company created by the Spanish breeders association (ANOVE) - we elaborated a database with the SSR genotypes of the varieties owned by its associates, particularly of fruit, berry and cut flower crops. This database, which was produced with leaf materials from the official repositories of these species, contained the standards with which we could compare any sample sent for analysis by GESLIVE. DNA fingerprinting data have been accepted in court as strong evidence of identity between a known variety and material suspected of having been reproduced without license. The alternative in the past were field trials where the cultivars were compared by their phenotype, a long and expensive operation that made the defense of breeder’s rights impractical. In peach, where isozyme variability was only capable of separating a few classes based on marker genotype, only 16 selected SSRs were able to identify virtually all materials. Individuals that were identical with markers were likely to be sports or duplications. The IRTAgen service, was created in 1999 to provide results from fingerprinting or other applications related with MAS to individuals, companies or the public administration. IRTAgen is still operative and has provided many thousands of analyses in almost every crop that is important in Spain. A change from SSRs to SNPs is currently underway to provide more reliable and cheaper tests for its clients. One of the key elements for analyzing the relationship between genotype and phenotype is the generation of the appropriate tools to maximize its efficiency. We have developed many progenies for map construction and major gene or QTL analysis, with the most efficient being the Near-Isogenic line (NIL) collections. These consist of a set of plants with the genetic background of a certain elite genotype, each with a single DNA fragment from an exotic genotype. The sum of the exotic fragments of a NIL collection covers the whole genome of the exotic parent. Each NIL is an inbred line that can be reproduced by selfing, and any phenotypic difference when compared to the elite genotype can be attributed to the exotic DNA fragment. NILs are especially interesting for analyzing complex quantitative characters, allowing the exploration of the variability brought by exotic germplasm or wild relatives, detecting more QTLs than other classical populations - in part because they can be identified in the homogeneous background of the elite genotype. This allows the Mendelization of these QTLs, providing materials to study QTL interaction (by crossing two NILs with two different QTLs

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Molecular markers for plant genetics and breeding

Rosaceae (apple, peach and strawberry) were sequenced in a short time, it was possible to compare their genomes and infer their evolution from a common ancestral genome (Illa et al., 2011). The founder members of CRAG (CSIC and IRTA) lead the project for melon, along with private breeding companies, and public, Spanish and Autonomic, support. Using the shotgun approach with 454 sequencing technology, the 450 Mb genome was assembled and a set of more than 37,000 protein-coding genes annotated (Garcia-Mas et al. 2012). We have also been involved in the sequencing of the bean genome (Vlasova et al. 2016), and are currently in the consortia that are sequencing the genomes of octoploid strawberry and almond.

of vegetables. This research, partly supported by the Miquel Agustí Foundation (www.fundaciomiquelagusti.com), has been focused on beans, tomato, cabbage and onion. Markers have been used to genetically characterize these materials (Sánchez et al. 2007; Casals et al. 2011; Simó et al. 2014) and to study the inheritance of characters of interest for germplasm conservation (Casañas et al. 2013).

Conclusion More than 40 years after first being used in plant genetics, molecular markers have become a necessary tool for genetic analysis, being essential for understanding the inheritance of complex characters, identifying genes responsible for these characters and in the search for novel variation. Their use is widespread in plant improvement programs where they have been incorporated in the three essential phases of the classical breeding methods: identification of adequate parents for crosses, selection in segregating progenies, and quality control of seeds or plants. The role of markers as predictors of the phenotype has been enhanced by the use of whole-genome approaches such as genomic selection, where important gains are expected in the years to come. Catalan groups have pioneered some of the research in molecular markers internationally, particularly in fruit trees and vegetable crops, where they have built strong and lasting collaborations with the private sector. This research is currently very active and, provided that public support is adequate, promises to continue producing good results and scientific leadership over the next few decades.

Research on molecular markers in cereals at IRTA, University of Lleida (UdL) and University of Barcelona (UB) Breeding programs on durum wheat, barley, bread wheat and triticale were established in Catalonia at the beginning of the 1980’s. Research on these species, particularly bread wheat and barley, was done in collaboration between IRTA and the UdL, with occasional participation of the UB. Markers were identified in these species as useful tools for germplasm management and variability analysis (Royo et al. 2010; Soriano et al. 2016), and to understand crop evolution (Molina-Cano et al. 1999; Igartua et al. 2015) with the aim of identifying useful variability for breeding. Additionally, markers have been used for the genetic analysis of complex characters (Giraldo et al. 2016), particularly those related with drought resistance (Comadran et al. 2008; Maccaferri et al. 2011), the identification of chromosomal rearrangements (Farré et al. 2012), and the location of important genes such as pest resistance (Martín-Sánchez et al. 2003). In spite of their high economic importance and the enormous international research that makes this group of species one of the best characterized at the genetic level, the genomes of cereals have been among the last to be sequenced due to their large size, so requiring massive economic and technological effort. Once this important development is complete, coupled with the tools adequate for whole genome analysis at affordable prices, a bright future for marker-based strategies to foster the improvement of this important group of crops is expected.

Acknowledgments. Thanks are indebted to many researchers that have made possible the results described here from the Departments of Plant Genetics, and Genomics and Biotechnology of IRTA; among others J. Garcia-Mas, A. Monfort, W. Howad, M.J. Aranzana, M. Martín, M. Pujol, M. Mnejja, K. Alexiou and I. Eduardo. The public and private support received by IRTA is acknowledged, especially that received from three companies with which we have collaborated for more than 20 years: Semillas Fitó S.A., Plantas de Navarra S.A. and Rústicas del Guadalquivir S.L.

References 1. 2.

Research on molecular markers at the Polytechnic University of Barcelona (UPC) A research line of the Department of Agrifood Engineering and Biotechnology at UPC is the collection, conservation, genetic analysis and sensory characterization of germplasm of locally important landraces and cultivars

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3. 4.

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Ahn S, Tanksley SD (1993) Comparative linkage maps of the rice and maize genomes. Proc Nat Acad Sci USA 90:7980-7984 Bernatzky R, Tanksley SD (1986) Toward a saturated linkage map in tomato based on isozymes and random cDNA sequences. Genetics 112:887-898 Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314-331 Casals J, Pascual L, Canizares J, Cebolla-Cornejo J, Casañas F, Nuez F (2011) The risks of success in quality vegetable markets: Possible genetic erosion in Marmande tomatoes and consumer dissatisfaction. Sci Hortic 130: 78-84

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24. Oliver M, Garcia-Mas J, Cardús M, Pueyo N, López-Sesé AI, Arroyo M, Gómez-Paniagua H, Arús P, de Vicente MC (2001) Construction of a reference linkage map for melon. Genome 44:836-845 25. Paterson AH, lander ES, Hewitt JD, Peterson S, Lincoln SE, Tanksley SD (1988) Resolution of quantitative traits into Mendelian factors by using a complete linkage map of restriction fragment lenght polymorphisms. Nature 335:721-726 26. Royo C et al. (2010) Understanding the relationships between genetic and phenotypic structures of a collection of elite durum wheat accessions. Field Crops Res 119: 91-105 27. Sánchez E, Sifres A, Casañas F, Nuez F (2007) Common bean (Phaseolus vulgaris L.) landraces in Catalonia a Mesoamerican germplasm hotspot to be preserved. J Hortic Sci Biotechnol 82: 529-534 28. Serra O, Donoso JM, Picañol R, Batlle I, Howad W, Eduardo I, Arús P (2016) Marker-assisted introgression (MAI) of almond genes into the peach background: a fast method to mine and integrate novel variation from exotic sources in long intergeneration species. Tree Genet Genomes 12:96 29. Shulaev V et al. (2011) The genome of woodland strawberry (Fragaria vesca). Nature Genetics 43:109-116 30. Simó J, Pascual L, Cañizares J, Casañas F (2014) Spanish onion landraces (Allium cepa L.) as sources of germplasm for breeding cal double dagger ots: a morphological and molecular survey. Euphytica 195:287-300 31. Soriano JM, Villegas D, Aranzana MJ, García del Moral LF, Royo C (2016) Genetic structure of modern durum wheat cultivars and Mediterranean landraces matches with their agronomic performance. PLOS ONE 11:e0160983    32. Tanksley SD, Medina-Filho H, Rick CM (1981) The effect of isozyme selection on metric characteris in an interspecific backcross of tomato - basis of an early screening procedure. Theor Appl Genet 60:291296 33. Tanksley SD, Orton TJ (1982) Isozymes in plant genetics and breeding. Part A and B. Elsevier Science Publishers BV, Amsterdam, The Netherlands 34. Tanksley SD, Medina-Filho H, Rick CM (1982) Use of naturally-occurring enzyme variation to detect and map genes controlling quantitative traits in an interspecific backcross of tomato. Theor Appl Genet 49:11-25 35. Tanksley SD, Young ND, Paterson AH, Bonierbale MW (1989) RFLP mapping in plant breeding: new tools for an old science. BioTechnology 7:257-263 36. Urrutia M, Bonet J, Arús P, Monfort (2015a) A near-isogenic line (NIL) collection in diploid strawberry and its use in the genetic analysis of morphologic, phenological and nutritional characters. Theor Appl Genet 128:1261-1275 37. Urrutia M, Schwab W, Hoffmann T, Monfort A (2015b) Genetic dissection of the (poly)phenol profile of diploid strawberry (Fragaria vesca) fruits using a NIL collection. Plant Sci. 242:151-168 38. Verde I et al. (2013) The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nature Genet 45:487-494 39. Viruel M, Messeguer R, de Vicente MC, Garcia-Mas J, Puigdomènech P, Vargas F, Arús P (1995) A molecular marker map with RFLPs and isozymes for almond. Theor Appl Genet 91:964-971 40. Vlasova A et al. (2016) Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes. Genom Biol 17:32.

Casañas F, Pérez-Vega E, Almirall A, Plans M, Sabaté J, Ferreira JJ (2013) Mapping of QTL associated with seed chemical content in a RIL population of common bean (Phaseolus vulgaris L.) Euphytica 192:279-288   Comadran J et al. (2008) Mapping adaptation of barley to droughted environments. Euphytica 161:35-45    Dirlewanger E, Graziano E, Joobeur T, Garriga-Calderé F, Cosson P, Howad W, Arús P (2004) Comparative mapping and marker-assisted selection in Rosaceae fruit crops. Proc Nat Acad Sci USA 101:9891-9896 Eduardo I, Arús P, Monforte AJ (2005) Development of a genomic library of near isogenic lines (NILs) in melon (Cucumis melo L.) from the exotic accession PI161375. Theor Appl Genet 112:139-148 Farré A, Cuadrado A, Lacasa-Benito I, Cistué L, Schubert I, Comadran J, Jansen J, Romagosa I (2012) Genetic characterization of a reciprocal translocation present in a widely grown  barley  variety. Mol Breeding 30: 1109-1119 Illa E, et al. (2011) Comparative analysis of rosaceous genomes and the reconstruction of a putative ancestral genome for the family. BMC Evolutionary Biology 11:9 Joobeur T, Viruel MA, Vicente MC, Jáuregui B, Ballester J, Dettori MT, Verde I, Truco MJ, Messeguer R, Batlle I, Quarta R, Dirlewanger E, Arús P (1998) Construction of a saturated linkage map for Prunus using an almond×peach F2 progeny. Theor Appl Genet 97:1034-1041 Garcia-Mas J et al. (2012) The genome of melon (Cucumis melo L.), Proc Natl Acad Sci USA. 109(29):11872-11877 Giraldo P, Royo C, Gonzalez M, Carrillo JM, Ruiz M (2016) Genetic diversity and association mapping for agromorfological and grain quality traits of a structured collection of durum wheat landraces including subsp. durum, turgidum and diccocon. PLoS ONE, 11(11), e0166577. Gottlieb LD (1982) Conservation and duplication of isozymes in plants. Science 216:373-379 Gottlieb LD (1981) Electrophoretic evidence and plant populations. Phytochemistry 7:1-45 Igartua E et al. (2015) Selection footprints in barley breeding lines detected by combining genotyping-by-sequencing with reference genome information Mol Breeding 35:11 Howad W, T. Yamamoto, E. Dirlewanger, R. Testolin, P. Cosson, G. Cipriani, A. J. Monforte, L. Georgi, A.G. Abbott and P. Arús. 2005. Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map. Genetics 171:1305-1309 Li XW, Meng XQ, Jia HJ, Yu ML, Ma RJ, Wang LR, Cao K, Shen ZJ, Niu L, Tian JB, Chen MJ, Xie M, Arús P, Gao ZS, Aranzana MJ (2013) Peach genetic resources: diversity, population structure and linkage disequilibrium. BMC Genetics 14:84 Maccaferri M et al. (2011) Association mapping in durum wheat grown across a broad range of water regimes. J Exp Bot 62: 409-438 Martín-Sánchez JA et al. (2003) A new Hessian fly resistance gene (H30) transferred from the wild grass Aegilops triuncialis to hexaploid wheat. Theor Appl Genet 106:1248-1255 Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci 88:1212-1217 Molina-Cano JL, Moralejo M, Igartua E, Romagosa I (1999) Further evidence supporting Morocco as a centre of origin of barley. Theor Appl Genet  98 :913-918 Nieto C et al. (2006) An eIF4E allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon. Plant J 48: 452-462

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RESEARCH REVIEWS O P E N A ACCE S S

CONTRIB SCI 13(1):17-32 (2017 ) doi:10.2436/20.7010.01.257

Institut d’Estudis Catalans, Barcelona, Catalonia

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HDL function and novel HDL-targeted therapies for preventing atherosclerotic cardiovascular disease: From mouse models to human disease Marina Canyelles1 , Lídia Cedó2-3, Josep Julve2-4 , Joan Carles Escolà-Gil2-4, Francisco Blanco-Vaca1-4 � Servei Bioquímica Clínca, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. � Institut d’Investigacions Biomèdiques (IIB) Sant Pau, Barcelona, Spain. � CIBER de Diabetes y Enfermedades Metabólicas Asociadas, CIBERDEM, Hospitalet de Llobregat, Spain. � Departament de Bioquímica, Biología Molecular i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain.

Resum. Malgrat que la relació inversa entre les concentracions baixes de colesterol associat a lipoproteïnes d’alta densitat i l’increment del risc de patir una malaltia cardiovascular està comunament acceptada, l’ús d’alguns fàrmacs que incrementen les concentracions de colesterol d’HDL no s’ha trobat associat a una reducció en l’aparició d’episodis cardiovasculars. Es considera que el transport revers de colesterol (RCT) és el principal responsable de l’efecte cardioprotector de l’HDL, estimulant el flux de colesterol des dels macròfags de les cèl·lules escumoses en la lesió arterioscleròtica cap a fetge on és eliminat parcialment en femtes. A més del RCT, les HDL tenen altres propietats cardioprotectores degudes als seus efectes antioxidant i antiinflamatori. Els estudis en ratolins modificats genèticament han permès demostrar que aquestes funcions de les HDL estan associades a la susceptibilitat a l’arterioesclerosi i suggereixen que fàrmacs que les puguin estimular podrien ser considerats com a noves estratègies terapèutiques per a prevenir el desenvolupament de la placa arterioescleròtica i el risc d’episodis cardiovasculars. El potencial antitumorogènic de les HDL és una àrea d’investigació recent. Aquests tipus d’investigacions són essencials per a poder traslladar els resultats obtinguts a humans.

Els estudis més recents indiquen que l’augment del flux de colesterol des dels macròfags fins a les HDL, el primer pas del RCT, està inversament relacionat amb l’aparició d’episodis cardiovasculars. Per tant, la majoria d’estudis actuals van enfocats a millorar les propietats cardioprotectores de les HDL, més que no pas a intentar augmentar les concentracions de colesterol d’HDL. Abstract. Although significant evidence supports the concept that low high density lipoprotein cholesterol (HDL-C) is associated with an increased risk of cardiovascular disease (CVD), the failure of several HDL-targeted therapies to reduce CVD has cast doubts on the HDL-C hypothesis. Reverse cholesterol transport (RCT) is currently thought to be a major HDL cardioprotective property by which HDL promotes cholesterol efflux from macrophage foam cells and delivers that cholesterol to the liver, from where it will be partly eliminated through bile and feces. Beyond RCT, HDL exhibits other cardioprotective properties, such as antioxidant and anti-inflammatory effects. Data from genetically-engineered mice indicate that these HDL functions are closely associated with atherosclerosis susceptibility, thereby suggesting that the promotion of HDL

Keywords: HDL, cardiovascular disease, CETP, apoA-I, ATP-binding cassette, reverse cholesterol transport, nuclear receptors Correspondence:

Dr. Francisco Blanco-Vaca fblancova@santpau.cat

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HDL function and novel HDL-targeted therapies for preventing atherosclerotic cardiovascular disease

functional properties may be considered a novel therapeutic strategy to reduce the atherosclerotic plaque burden and subsequent cardiovascular events. Furthermore, the potential anti-tumorigenic role of HDL is currently under investigation. This research has been essential for trying to translate experimental results obtained in mice to humans. The results of several recent human studies indicate that enhanced macrophage cholesterol efflux, the first step of RCT, is inversely associated with CVD events. Therefore, the current research is focused on improving HDL function rather than simply targeting HDL-C levels.

The failure of various HDL-targeted therapies to reduce CVD has cast doubts on the HDL hypothesis. Two large trials (ILLUMINATE and dal-OUTCOMES) with cholesteryl ester transfer protein (CETP) inhibitors were stopped for disappointing results or reason of futility (Barter and Rye, 2012), and a large Mendelian randomization study showed that a single-nucleotide polymorphism (SNP) in the endothelial lipase (EL) gene did not affect the risk of myocardial infarction despite increasing HDL cholesterol (HDL-C) (Voight et al., 2012). Therefore, the inverse relation between cardiovascular risk and HDL is no strictly related to the mass of cholesterol transported by HDL (Rader and Hovingh, 2014). HDL comprises a complex mixture of heterogeneous lipoproteins ranging from nascent preβ-HDL particles to mature HDL spherical particles. Apolipoprotein (apo) A-I is the main HDL protein and significant evidence reveals that most HDL antiatherogenic properties depend on apoA-I content (Mineo and Shaul, 2012). Among these anti-atherosclerotic properties, the best recognized is the ability of HDL to stimulate efflux of excess cholesterol from macrophage foam cells, and consequently this is the most evaluated mechanism (Camont et al., 2011). This efflux to HDL occurs via different pathways, the most efficient ones being mediated by two cholesterol transporters, transmembrane ATP-binding cassette (ABC): ABCA1 and ABCG1. ABCA1 promotes cholesterol transport to nascent preβ-HDL and lipid free apoA-I, whereas ABCG1 facilitates the efflux to mature α-migrating HDL. Macrophage ABCA1 and ABCG1 form the primary route of cholesterol efflux and, therefore, start the macrophage-to-feces RCT (Figure 1). The results of several recent studies indicate that stimulation of macrophage cholesterol efflux to HDL mediated by ABCA1 transporter is inversely associated with the incidence of atherosclerotic CVD events, thereby indicating the importance of this function as biomarker for predicting CVD risk (Rohatgi et al., 2014; Saleheen et al., 2015). The efflux of cellular cholesterol to HDL initiates RCT in all tissues, but the fraction that originates from the macrophage foam cells located in the intima is considered the main RCT component directly involved in atherosclerosis (Cuchel and Rader, 2006). An assay for measuring in vivo macrophage-to-feces RCT by tracing the reverse [3H]-cholesterol transport from lipid-laden macrophages to feces in mice was developed (Rader et al., 2009). The method has been used to study the role of different therapies and pathways relevant for RCT and HDL-mediated atheroprotection (Escola-Gil et al., 2009; Annema and Tietge, 2012). However, this method is only applicable in animal models and, for ethical issues, seems impossible to apply in hu-

Abbreviations. HDL: high density lipoprotein; LDL: low density lipoprotein; CVD: cardiovascular disease; CETP: cholesteryl ester transfer protein; ACAT: cholesterol acyl transferase; PLTP: phospholipid transfer protein; HL: hepatic lipase; EL: endothelial lipase; ApoA-I: apolipoprotein A-I; ABC: ATP-binding cassette; RCT: reverse cholesterol transport; LXR: liver X receptor; PPAR: peroxisome proliferator-activated receptor; NPC1L1: niemann-Pick C1-Like 1; CYP7A1: cytochrome P450 family 7 subfamily 1; PON1: paraoxonase/arylesterase 1; PAF-AH: platelet-activating factor-acetylhydrolase; SIRT: NAD+-dependent deacetylase sirtuin; SERM: selective estrogen receptor modulators; SFA: saturated fatty acids; PUFA: polyunsaturated fatty acids; TAM: tamoxifen; RAL: raloxifene; TOR: toremifene; ER: estrogen receptor; HPA: hypothalamic-pituitary-adrenal; CRF: corticotrophin releasing factor; CORT: corticosterone

Introduction The current concept that high-density lipoprotein (HDL) protects against cardiovascular disease (CVD) is based on abundant preclinical and epidemiological data. Originally, the main explanation was associated with the ability of HDL to induce cholesterol efflux from macrophage foam cells in atherosclerotic lesions (Glomset, 1968). Historically, the macrophage cholesterol efflux has been considered the main HDL antiatherogenic function. However, reverse cholesterol transport (RCT) is a complex pathway and macrophage cholesterol efflux is only considered the first step of the pathway. This antiatherogenic pathway may be regulated at different steps, such as (i) the macrophage cholesterol efflux, (ii) the transport of cholesterol through the plasma compartment, (iii) the liver uptake, (iv) the hepatobiliary excretion, and (v) the excretion from the body (see Figure 1 for details).

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Figure 1 legend. Schematic diagram of reverse cholesterol transport pathway. ApoA-I is synthesized by liver and small intestine, acquires phospholipids and become nascent partially lipidated preβ-HDL particles. Preβ-HDL particles acquire free cholesterol from macrophages via adenosine triphosphate-binding cassette (ABC) A1 transporter. Free cholesterol is converted into cholesteryl ester within the HDL particle by the action of lecithin:cholesterol acyltransferase (LCAT), thereby resulting in mature HDL. ApoA-I is the major HDL protein and activates LCAT, whereas apoA-II, the second HDL protein, displaces apoA-I form HDL particles. The scavenger receptor type BI (SR-BI) and ABCG1 facilitate the cholesterol efflux process from macrophages to mature HDL. Phospholipid transfer protein (PLTP) promotes the transfer of phospholipids and free cholesterol from triglyceride-rich lipoproteins into HDL, producing a remodelling process by which preβ-HDL particles can be generated. Furthermore, PLTP may promote the fusion of smaller HDL particles and subsequent generation of larger HDL particles. Endothelial lipase (EL) and hepatic lipase (HL) hydrolyse HDL triglycerides and phospholipids, thereby converting large HDL particles to smaller ones. HDL-cholesteryl ester can be transferred to VLDL or LDL by cholesteryl ester transfer protein (CETP) and returned to the liver through low-density lipoprotein receptor (LDLR) or other receptors. The liver also selectively takes up HDL-associated cholesterol via SR-BI. The liver ABCG5/G8 heterodimer is involved in cholesterol transport to bile. Niemann-Pick C1-like 1 (NPC1L1) is of crucial importance for absorbing macrophage-derived cholesterol in the small intestine. Cholesterol may also be excreted back to the lumen by the intestinal ABCG5/G8 heterodimer.

mans. Some studies are in process to discover the way to evaluate the entire RCT pathway in humans such as the use of [3H]-cholesterol/albumin complexes or [2,3-13C2]-cholesterol but the application of these assays are still awaited (Schwartz et al., 2004; Turner et al., 2012).

rosclerosis can be related to its stimulatory effect on macrophage-to-feces RCT, which has been demonstrated both in transgenic mice expressing human apoA-I (hApoA-I) (Zhang et al., 2003) and in wild-type mice intraperitoneally administered with a dose of hApoA-I (Lee-Rueckert et al., 2011). The generation of genetically-modified mice for ABCA1, apoA-II, CETP, hepatic lipase (HL) and EL has permitted to identify potential molecular targets for modulating macrophage-specific RCT and HDL antioxidant activity (see Table 1 for a summary).

HDL targets With the creation and detailed analysis of genetically-modified mice, a solid body of new information emerged on the mechanisms controlling the RCT pathway. Indeed, the atheroprotective role of apoA-I has been corroborated in transgenic mice (Rubin et al., 1991). Such beneficial effects of apoA-I on the development of athe-

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ATP-binding cassette transporter A1 (ABCA1) ABCA1 is present in liver, intestine and others peripheral tissues, particularly in macrophages. ABCA1 is required

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HDL antioxidant potential

HDL-C ABCA1 deficiency (Calpe-Berdiel et al., 2005) Human Apo A-II overexpression (Ribas et al., 2004; Rotllan et al., 2005)

Non determined

CETP overexpression (Rotllan et al., 2008)

=

HL and EL overexpression (Escola-Gil et al., 2013b)

Macrophage-to-feces RCT

=

=

Table 1. Assays of HDL function in genetically-modified mice.

apoA-II (hApoA-II) transgenic HDL to protect against oxidative modification of apoB-containing lipoproteins (Escola-Gil et al., 2000). We found a significant increase in the amount of aortic antigens related to LDL oxidation in transgenic mice overexpressing hApoA-II. HDL of transgenic mice failed to protect the apoB-containing lipoproteins from oxidation, including very low-density lipoprotein (VLDL) and LDL. Human apoA-II-containing HDL also showed a decreased content of the main HDL antioxidant enzymes paraoxonase/arylesterase 1 (PON1) and platelet-activating factor-acetylhydrolase (PAF-AH). Incubating isolated hApoA-II with control plasma at 37ºC decreased PON1 activity in plasma and HDL. The displacement of PON1 by physiologic concentrations of hApoAII could explain why PON1 is mostly found in HDL particles without apoA-II, as well as, the lack of antiatherogenic properties of apoA-II-enriched HDL (Ribas et al., 2004). In an independent study, we investigated the effects of hApoA-II on the entire RCT pathway by using transgenic mice under chow or an atherogenic diet. On the chow diet, hApoA-II overexpression accelerated the transfer of macrophage-derived cholesterol to liver and feces. However, the magnitude of macrophage-to-feces RCT did not differ between transgenic and control mice fed the atherogenic diet. Taken together, our results indicate that hApoA-II exerts its proatherogenic effect by counteracting antioxidant properties of HDL rather than by impairing macrophage-to-feces RCT (Escola-Gil et al., 2000; Ribas et al., 2004; Rotllan et al., 2005).

for the maintenance of plasma HDL levels via preβ-HDL formation (Figure 1), which are among the main acceptors of cholesterol efflux (Basso et al., 2003). Macrophage-selective inactivation of ABCA1 promotes atherosclerotic lesions without affecting plasma HDL levels (Aiello et al., 2002; Francone et al., 2005). We investigated the in vivo role of ABCA1 in macrophage-specific RCT by using the wild-type ABCA1+/+, ABCA1+/− and ABCA1-deficient (ABCA1−/−) mice (Calpe-Berdiel et al., 2005). Whole body cholesterol balance did not differ among genotypes, indicating that total RCT does not seem to be affected by ABCA1 or HDL deficiency. However, a direct relationship was observed between ABCA1 gene dose and the amount of macrophage-derived [3H]-cholesterol in plasma. Importantly, ABCA1−/− mice had a significantly reduced excretion of fecal [3H]-cholesterol (Calpe-Berdiel et al., 2005). In line with these findings, selective inactivation of ABCA1 in macrophages also impaired macrophage-to-feces RCT (Wang et al., 2007). Overall, these findings confirm the crucial role of ABCA1 in this atheropretective mechanism of HDL (Lee-Rueckert et al., 2016). Apolipoprotein A-II In contrast with the cardioprotective effect of apoA-I, the overexpression of mouse and human apoA-II has usually been found to be proatherogenic (Warden et al., 1993; Escola-Gil et al., 1998). However, the role of apoA-II on major HDL functions remains unclear. One of the mechanisms by which apoA-II cause atherosclerosis may be an impaired ability of HDL to protect against low density lipoprotein (LDL) oxidation, as reported in mouse apoA-II transgenic mice (Castellani et al., 1997). We investigated the potential of human

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Cholesteryl ester transfer protein (CETP) CETP promotes the removal of cholesteryl ester from HDL in heteroexchange for triglycerides derived from LDL,

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VLDL or chylomicrons (Figure 1). CETP transgenic mice, which naturally do not have this transporter protein, showed increased susceptibility to atherosclerosis, although the effects of CETP on CVD in human studies remain unclear (Tall, 1993; de Grooth et al., 2004). We determined the influence of CETP activity on the two major antiatherogenic functions of HDL by using transgenic mice overexpressing CETP. The magnitude of macrophage-derived cholesterol in liver and feces did not differ between CETP transgenic mice and control mice and this was independent of the diet used (chow or atherogenic). Furthermore, the injection of endogenous CETP-expressing macrophages did not alter macrophage RCT in control mice. HDL from CETP-expressing mice protected LDL from oxidative modification at similar levels that did the HDL from control mice (Rotllan et al., 2008). Although we did not find significant evidence that CETP affected these major HDL functions, adenovirus-mediated human CETP expression promoted the macrophage-dependent RCT rate (Tanigawa et al., 2007; Tchoua et al., 2008). In any event, these results would not predict the increased atherosclerosis susceptibility of CETP transgenic mice.

lipase enzyme while HL has phospholipase and triglyceride lipase activities; both enzymes may convert large HDL to smaller HDL (Yasuda et al., 2010). HL- and EL-deficiency in mice caused a significant increase on HDL lipid and apoA-I levels (Ishida et al., 2003). A physiologically important question is whether HL and EL activities alter HDL cardioprotective functions. We evaluated the effects of HL and EL deficiency on macrophage-to-feces RCT pathway in vivo, the susceptibility to oxidation of HDL, and its ability to protect against LDL oxidation (Escola-Gil et al., 2013b). As expected, both HL- and EL-deficiency caused an increase in cholesterol and phospholipids associated to HDL and an increase in HDL size. ApoA-I and PAF-AH were also increased in both HL- and EL-deficient mice but no changes in PON1 activity were found. These changes did not correlate with alterations in the liver expression of enzymes involved in HDL metabolism. These genetically-modified mice displayed increased levels of macrophage-derived HDL-bound [3H]-cholesterol which was concomitant with increased levels of [3H]-cholesterol in feces. HDL from the HL- and EL-deficient mice was less prone to oxidation and had a higher ability to protect LDL from oxidation. These changes were more pronounced in the EL-deficient mice (Escola-Gil et al., 2013b). However, the role of HL- and EL-deficiency on atherosclerosis development provided divergent data

Hepatic lipase (HL) and endothelial lipase (EL) HL and EL are part of the triglyceride lipase family and differ in their hydrolytic activities. EL is mainly a phospho-

Drug Fenofibrate Fibrates (Rotllan et al., 2011) Gemfibrozil

LXR agonist (Calpe-Berdiel et al., 2008)

T0901317

Tamoxifen SERMs (Fernandez-Suarez et al., 2016)

Raloxifen Ezetimibe

Cholesterol absorbtion (Silvennoinen et al., 2012) PPARδ agonist GW0742

HDL antioxidant potential

Macrophage-to-feces RCT

Human apoAI, PLTP and liver ABCG5/G8 ↑

=

Liver ABCG5/G8 ↑

=

Liver ABCA1, ABCG5/G8 and ABCG1 ↑ and intestine ABCA1 and ABCG1 ↑

Liver ABCG5/G8 ↓

HDL-C

↓ ↓

=

=

Intestinal NPL1C1 ↓

=

Intestinal NPL1C1 ↓

Table 2. Effects of therapeutic agents on macrophage-to-feces RCT.

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the amount of nascent preβ-HDL particles and plasma phospholipid transfer protein (PLTP) activity, concomitant with an upregulation of Pltp and human APOA1 gene expression. In contrast, gemfibrozil did not affect these plasma parameters and the macrophage-derived [3H]-cholesterol flux to plasma and feces of hApoA·l transgenic mice. Unlike gemfibrozil, fenofibrate also induced the generation of larger HDL particles, which were more enriched in cholesteryl esters, and promoted macrophage cholesterol efflux to plasma in vitro. None of the drugs affected net intestinal cholesterol absorption. These findings demonstrated that fenofibrate, but not gemfibrozil, promoted in vivo macrophage-specific RCT, thereby highlighting the differential action of this fibrate on HDL functionality. These data would be consistent with a protective clinical effect of fenofibrate that remains to be clearly demonstrated in interventional human studies (Rotllan et al., 2011).

since other HDL-independent biological properties of HL and EL, such as their role in modulating apoB-containing lipoprotein catabolism, macrophage cholesterol uptake and inflammation influence (Annema and Tietge, 2011).

Pharmacology RCT-enhancing therapies are currently considered a promising strategy for the prevention and treatment of atherosclerotic CVD. An important number of RCT-targeted drugs have been used in mice to test their effects on macrophage-specific RCT in vivo. Some of these therapeutic approaches included fibrates, liver X receptor (LXR) activation and selective estrogen receptor modulators (SERMs), and cholesterol absorption inhibitors (Table 2). Fibrates Fibrates are peroxisome proliferator-activated receptors alpha (PPARα) agonists commonly used mainly in hypertriglyceridemic patients. Clinical effects of fibrates are attributed to their ability to reduce triglycerides and increase HDL-C levels (Fruchart et al., 1999; Staels et al., 2008). Current guidelines recommend the use of fenofibrate for preventing cardiovascular disease, particularly for high-risk statin-treated patients with atherogenic dyslipidemia. Furthermore, fenofibrate appears to exert a stronger effect on plasma hApoA-I levels that other fibrates. However, evidence that fibrates reduce mortality and morbidity associated with CVD is still unclear (Grundy et al., 2004; Duez et al., 2005). Since HDL metabolism is regulated in an opposite manner by fibrates in wild-type rodents due to sequence divergences in the mouse Apoa1 promoter, our group investigated the effects of fibrates on the entire macrophage-dependent RCT pathway in mice overexpressing hApoA-I, a model that elicits a humanized response to fibrates (Rotllan et al., 2011). We used hApoA-I transgenic mice treated with gemfibrozil, fenofibrate or vehicle as control group. We found that fenofibrate increased significantly macrophage-derived plasma [3H]-cholesterol 24 and 48 hours after macrophage injection and the net fecal [3H]-cholesterol + bile acid excretion over 48h. Both fibrates increased mRNA levels of liver Ppara and two key liver genes involved in hepatic cholesterol transport to bile and feces, Abcg5 and Abcg8; but only fenofibrate induced lipid profile modifications. After fenofibrate treatment, total plasma hApoA-I and HDL-C levels in female transgenic mice were higher than those of vehicle-treated mice. Fenofibrate also caused an increase in

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Liver X activation The LXR α and β are oxysterol-activated nuclear receptors that regulate the expression of a number of genes involved in RCT such as ABCA1, ABCG1, ABCG5, ABCG8 and cytochrome P450 7a1 (CYP7A1) (Repa et al., 2002). LXRα is expressed mainly in liver and secondarily in adrenal glands, intestine, adipose tissue, macrophages, lung and kidney. LXRβ is expressed in almost all tissues (Zelcer and Tontonoz, 2006). Overexpression of ABCG5/G8 or activation of LXRαβ results in increased hepatobiliary excretion of cholesterol and increased fecal neutral sterol excretion (Yu et al., 2002a). Consistent with these findings, the inactivation of these two genes has opposite effects on sterol hepatobiliary excretion (Yu et al., 2002b). Synthetic high-affinity LXRαβ agonists promote RCT from macrophages to feces in vivo despite having a limited impact on HDL-C levels. These specific activators also inhibit atherosclerosis progression, and even promote atherosclerosis regression in mice (Terasaka et al., 2003). The results of these studies support the concept that LXR activation promotes macrophage-specific RCT pathway by increasing biliary cholesterol excretion through ABCG5/G8 upregulation (Yu et al., 2003). We initially investigated the effects of ABCG5/G8 deficiency as well as those of LXR agonist induction of RCT from mouse macrophages to feces in ABCG5/G8-deficient (ABCG5/G8-/-) and wild-type (ABCG5/G8+/+) mice (Calpe-Berdiel et al., 2008). When [3H]-cholesterol-labeled mouse macrophages were injected intraperitoneally into the ABCG5/G8+/+ and ABCG5/G8-/- mice, we found an in-

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of SERMs are tamoxifen (TAM) and toremifene (TOR), which are mainly used in breast cancer, and raloxifene (RAL), which is used for the treatment of osteoporosis in postmenopausal women (Pickar et al., 2010). Beyond their therapeutics effects, SERMs also reduce cholesterol associated to LDL-C. This effect may be mediated by the decrease of cholesterol biosynthesis, the increase of the expression of LDL receptor (LDLR), and the inhibition of cholesterol acyl transferase (ACAT) (Suarez et al., 2004; Cerrato et al., 2015). We have investigated whether the SERM-mediated interference of cholesterol trafficking in macrophages may restrict cholesterol efflux to HDL and, consequently, impair the cholesterol transport to feces in vivo. As expected, SERMs impaired intracellular acetylated LDL (acLDL)-derived cholesterol trafficking and cholesterol efflux in macrophages (Fernandez-Suarez et al., 2016). Human THP-1 macrophages were loaded with [3H]-cholesterol-labeled acLDL in the presence of SERMs and cholesterol efflux to different acceptors was determined. TAM decreased apoA-I-mediated cholesterol export in a dose-dependent manner, and HDL-mediated cholesterol efflux was also reduced. RAL and TOR also decreased apoA-I-mediated cholesterol efflux, but not the HDL-mediated cholesterol efflux. AcLDL markedly increased the protein levels of ABCA1 and ABCG1 in macrophages, whereas TAM, RAL and TOR had the opposite effect. The TAM-mediated effect on macrophage [3H]-cholesterol efflux was not related with its effects on cholesterol intracellular trafficking. Therefore, SERMs impaired cholesterol export from THP-1 macrophages by two ways: lowering the availability of lipoprotein cholesterol and impairing the acLDL-mediated induction of ABCA1 and ABCG1. Moreover, SERMs effects were ER independent because ESR1 and ESR2 expression was undetectable in THP-1 macrophages and the addition of an ER down-regulator did not alter the effect of any SERM. These effects were also confirmed in human monocyte-derived macrophages. Importantly, when [3H]-cholesterol-loaded macrophages were intraperitoneally injected into mice, TAM, but not RAL, decreased the [3H]-cholesterol levels in serum, liver and feces. This effect was primarily attributable to the TAM-mediated reduction of the capacity of HDL to promote cholesterol mobilization from macrophages (Fernandez-Suarez et al., 2016).

creased radiolabeled HDL-bound [3H]-cholesterol after the label injection. However, not significant differences were found between the two groups in terms of [3H]-tracer in liver, feces and bile acids. These findings were rather surprising, considering that disruption of ABCG5/G8 resulted in a marked reduction in biliary cholesterol levels. This could indicate an important role of the intestine in the model used in this study. Recent data suggest that the intestine may play an important role in excreting cholesterol from plasma to the intestinal lumen and feces. Then, we investigated whether ABCG5/G8 were necessary for LXR-mediated induction of macrophage-specific RCT pathway by injecting the radiolabeled macrophages into the ABCG5/G8+/+ and ABCG5/G8-/- mice treated with or without the LXR agonist T0901317. Treatment with T0901317 increased liver ABCG5/ G8 expression, and was associated with a 2-fold increase in the fecal excretion of macrophage-derived [3H]-cholesterol of ABCG5/G8+/+ mice. However, the LXR treatment did not affect fecal [3H]-cholesterol excretion in ABCG5/G8-/- mice. We also determined the fate of [3H]-cholesterol from HDL by radiolabeling HDL with HDL-[3H]-cholesteryl oleate. Untreated and T0901317-treated ABCG5/G8+/+ and ABCG5/G8-/mice were injected intravenously with the radiolabeled HDL. Plasma clearance of intravenously injected [3H]-HDL was significantly slower in treated ABCG5/G8+/+ mice compared with untreated mice. However, LXR activation did markedly increase the recovery of HDL-derived [3H]-cholesterol in the feces of ABCG5/G8+/+ mice. Conversely, LXR agonists did not have the ability to increase fecal [3H]-cholesterol in ABCG5/ G8-/- mice (Calpe-Berdiel et al., 2008) Our results demonstrated that ABCG5/G8 transporters are essential for the LXR agonist-mediated induction of RCT from macrophages to feces in vivo, thereby suggesting that upregulation of ABCG5/G8 may be an effective therapeutic strategy to increase macrophage-to-feces RCT pathway and reduce atherosclerosis. However, LXR activators have undesired effects such as hepatic triglyceride accumulation. Since LXRβ activation may target intestine, promote macrophage RCT and prevent atherosclerosis, current efforts have focused on developing novel LXRβ-selective ligands that circumvent liver side effects. In contrast, somewhat surprisingly, ABCG5/G8 deficiency in the mouse did not affect RCT from macrophages.

Cholesterol absorption inhibitors Both macrophage- and non-macrophage-derived cholesterol molecules are transferred through multiple steps of the reverse pathway to the intestine. On average, 50% of the cholesterol present in the intestinal lumen may be reabsor-

Selective estrogen receptor modulators (SERMs) SERMs are non-steroidal molecules that can be estrogen-agonists or antagonists depending on the tissue targeted and the kind of estrogen receptor (ER). Some examples

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bed, and the remainder will be ultimately excreted from the body as fecal cholesterol to complete the RCT pathway. The final regulatory steps of RCT that occur in the small intestine play an important role in determining the efficiency of cholesterol absorption and thus can influence the rate of the total body RCT (Lee-Rueckert et al., 2013). We have recently investigated the effects of modulating the traffic of cholesterol across the enterocyte on the rate of the specific itinerary of cholesterol from peripheral macrophages into feces. Therefore, interventions that inhibit cholesterol absorption including ezetimibe administration and PPARβ/δ activation with GW0742 increase the excretion of macrophage derived cholesterol in feces by reducing intestinal Niemann-Pick C1like protein 1 (NPC1L1) activity (Silvennoinen et al., 2012). Furthermore, certain pathophysiological conditions associated with an increased risk of atherosclerotic CVD have been found to modify intestinal cholesterol absorption and affect the macrophage-to-feces RCT pathway (see Physiological stress section for details).

There are certain physiopathological states that may affect HDL functionality such as stress or the immunological and inflammatory processes. HDL function has been also related with the potential anti-tumorigenic role of HDL.

CRF by CORT. When [3H]-cholesterol-loaded macrophages were injected into the mice, stress did not affect cholesterol efflux from macrophages but inhibited intestinal cholesterol absorption thus leading to accumulation of intestinal [3H]-cholesterol in the stressed mice. Furthermore, administration of CORT, the stress hormone in rodents, to non-stressed mice upregulated PPARα and downregulated NPC1L1 protein levels in the small intestine, fully reproducing the effect of stress on the rate of macrophage-to-intestine RCT in the non-stressed mice. The acceleration of RCT induced by stress was fully inhibited in mice pretreated with a cytochrome P450 inhibitor, metyrapone. No additive increase of macrophage-to-intestine RCT rate occurred if the stress mice were treated with ezetimibe or the PPARδ agonist GW0742 that blocks NPC1L1 action in the intestine (Silvennoinen et al., 2012). Overall, the results strongly indicated a functional connection between acute stress and decreased function of intestinal NPC1L1 via a mechanism likely mediated by PPARα, which is known to negatively regulate NPC1L1 expression in mice. Determination the macrophage-to-intestine RCT rate in PPARα knockout (KO) and LXRα KO mice exposed to stress shows that mere elevation of CORT by stress was not sufficient to promote macrophage-to-intestine RCT. Of note, we observed that the increase in the fecal excretion of macrophage-derived cholesterol induced by stress in mice was maintained for 7 days of chronic exposure (Silvennoinen et al., 2012).

Physiological stress In humans, a relationship between stress and atherosclerosis has been found in some epidemiological studies under conditions that trigger different types of stress (Rozanski et al., 1999). Simulated stress conditions in mouse models are useful for search mechanisms implicated in atherosclerosis. Previously studies indicate that the dual role of stress on atherosclerosis depends on the type of stress: it can be accelerated or remain unaffected (Kumari et al., 2003; Bernberg et al., 2009). We studied the rate macrophage-to-intestine RCT in mice exposed to restraint stress which mimics psychological stress in humans (Silvennoinen et al., 2012). We initially compared the results of a control mouse group without stress and mice exposed to physical restraint stress for 3 hours. Activation of hypothalamic-pituitary-adrenal (HPA) axis is the endocrine hallmark of stress and produce corticotrophin releasing factor (CRF) and corticosterone (CORT). In the stress group, CRF peaked and then rapidly returned to basal levels, whereas CORT levels remained elevated in stress group due to inhibition of

Anti-tumorigenic role of HDL Low levels of HDL-C have been associated with high risk of breast cancer (Ni et al., 2015). However the relationship between apoA-I and breast cancer remain unclear (Borgquist et al., 2016). ApoA-I has anti-inflammatory, antioxidant or anti-apoptotic properties. Some studies have demonstrated that hApoA-I-containing HDL has potent anti-tumor activity in xenograft mouse models of ovarian cancer and mouse models of malignant melanoma and Lewis lung carcinoma. ApoA-I mimetic peptides, which mimic the distribution of the charge and structure of portions of apoA-I, are considered as potential therapeutic agents for preventing a variety of inflammation-related diseases, including cancer. Indeed, two reports have demonstrated that the apoA-I mimetic peptide 4F significantly reduce tumor growth in xenograft mouse models of ovarian and colon cancer (Van Lenten et al., 2009; Su et al., 2010). We have recently investigated the effect of hapoA-I overexpression or administrated 4F in a validated mouse mammary tumor virus (MMTV) polyoma middle T antigen

Physiological and physiopathological states

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The hApoA-I stimulus on RCT was completely blocked in 48/80-treated mice with competent mast cells. In mast cell-deficient mice this RCT stimulated by apoA-I is higher in 48/80-treated mice indicating that the impaired RCT rate in 48/80-treated wild type mice was mast cell-dependent. Pre-incubation hApoA-I with or without a mouse chymase and posterior injection to mouse confirmed that impaired RCT was a consequence of apoA-I proteolysis. Although the fragments of apoA-I in peritoneal fluid could not be identified due to their rapid clearance, the study allowed to validate the hypothesis that activation of mast cells causes apoA-I proteolysis, and as a result, there is a decrease in the RCT pathway (Lee-Rueckert et al., 2011). The concentration of lipoprotein particles in the interstitial fluid is the result of a dynamic balance between entry via the capillaries and the exit via lymphatic system (Miller et al., 2011). Skin and intimal layer of arteries are two key points for cholesterol accumulation and may share common pathogenic mechanisms. HDL is removed from interstitial fluids by the lymphatic system and it has been shown that variation in its flow may stimulate RCT (Martel et al., 2013). We have recently investigated whether an increased vascular permeability promotes RCT from skin. Therefore, we evaluated the effect of increased vascular permeability in the skin on the RCT rate from J774 foam cells injected into the dorsal subcutaneous layer of the skin (Kareinen et al., 2015). The increase of the subcutaneous levels of histamine promoted HDL influx to skin and accelerated the rate of RCT from skin to feces. The treatment with different histamine-receptor antagonists indicate that the stimulatory effect of histamine on RCT was mainly mediated by histamine H1 receptor. These results were also found when other vasodilator agents were injected, such as serotonin and bradykinin. Exogenous administration of HDL and histamine in apoA-I deficient mice evidenced that the increase of circulating HDL levels and triggering of local vascular permeability were both required to enhance macrophage RCT from subcutaneously located macrophages. Importantly, the degranulation of activated mast cells releases a number of vasoactive components, including histamine, which was able to stimulate endogenously the RCT pathway (Kareinen et al., 2015). This contrasts with our previous findings that demonstrated the mast cell activation cause impaired macrophage-to-intestine RCT when hapoA-I was intraperitoneallly injected to stimulate RCT (Lee-Rueckert et al., 2011). The role of mast cell activation may probably depend on the balance between vascular permeability and apoA-I proteolysis.

(PyMT) transgenic mouse model that spontaneously develop adenocarcinomas in the mammary gland. The mimetic peptide 4F increased tumor latency, decreased the mammary gland weight and total tumor burden compared with vehicle treatment. However, hapoA-I did not produce any effect on tumor onset and growth despite increasing HDL-C. The anti-tumorigenic effects of 4F were closely associated with its ability to reduce the oxidized (ox) LDL (Cedo et al., 2016). Overexpression of hApoA-I also reduced the formation of oxLDL. However, the levels of 27-hydroxycholesterol (27-HC), a metabolite that has been reported to promote tumor growth in mouse models of ER-positive breast cancer (Nelson et al., 2013), were higher both in the serum and mammary tissue of PyMT-hApoA-I mice compared to control mice. The expression of Cyp7b1, the main enzyme implicated in 27-HC catabolism, was downregulated in mammary tissue of hApoA-I mice. This raised 27-HC could counteract the HDL-mediated benefits on the oxLDL levels and tumor progression and explain that the apoA-I mimetic peptide 4F, but not increased apoA-I-containing HDL levels, inhibits tumor growth in mice with inherited breast cancer. Our data are consistent with the reported anti-tumorigenic activity of apoA-I mimetics in other type of cancers (Cedo et al., 2016). Immune response Mast cell activation and vascular permeability. Mast cells are a type of white blood cell that function as a “master regulator� of the immune system. They are present in connective tissues around the body and in the arterial intima; mast cells have been found closely to macrophage foam cells (Kaartinen et al., 1995). Mast cell activation lead to a degranulation and chymase contained in its cytoplasm was able to modify HDL particles in vitro and to inhibit macrophage cholesterol efflux (Lee-Rueckert and Kovanen, 2006). We investigated whether local activation of mast cells would attenuate cholesterol efflux from neighboring macrophage foam cells, thereby disrupting the entire macrophage-to-feces RCT pathway (Lee-Rueckert et al., 2011). First, we demonstrated that the injection of mast cell-degranulating compound 48/80 in the peritoneal cavity of mice was able to inactivate by proteolysis the activity of apoA-I as an ex vivo cholesterol acceptor. Then, we evaluated the effects of injecting 48/80 into the C57Bl/6J mice to induce peritoneal mast cell activation, hApoA-I to stimulate RCT, and [3H]-cholesterol labeled J774 macrophages for measurement of the rate of RCT. After 3 hours, [3H]-radioactivity was measured in the intestinal lumen.

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tably, a commensurate favourable up-regulation in the hepatic gene expression of these two transporters, and other known targets of LXRa, was also observed in obese patients subjected to bariatric surgery along with an improvement in their fatty liver phenotype. Additionally, reduced liver Abcg5/g8 protein abundance in db/db mice has been also partly explained by the participation of post-transcriptional mechanisms involving enhanced endoplasmic stress (Sabeva et al., 2009). Interestingly, the induction of LXR has been also reported to ameliorate ER stress induced by obesity and hepatic lipid accumulation in vivo (Rong et al., 2013); potentially, this might help in the partial restoration of liver ABCG5/ G8 protein abundance in LXR-treated db/db mice. Taking together, these data might support the potential of targeting LXR signalling as a strategy to improve RCT in diabesity.

Autoimmune arthritis The relationship between dyslipidemia and the incidence of CVD in patients affected by rheumatoid arthritis has been established but it is still the subject of intense debate (John and Kitas, 2012). ApoE and inflammatory immune response are inversely correlated; however, the mechanism involved in this process remains unclear (Postigo et al., 2011). We recently investigated the severity of one type of autoimmune arthritis, Collagen-II-autoimmune arthritis (CIA), in apoE-deficient mice. ApoE deficiency was associated with higher severity of CIA (Alvarez et al., 2016). Consistent with these findings, a significantly increase in the expression of arthritogenic Il1β, Tnfa and Il6 was observed in apoE-deficient mice, thereby indicating that partial or total apoE deficiency modifies the polarization of macrophages after a potent inflammatory insult. ApoE-deficiency also altered the biochemical composition of HDL particles. However, the lipoprotein particles showed similar antioxidant ability, suggesting that the exacerbation of arthritis is largely independent of HDL function. We analyzed the role of hypercholesterolemia in the severity of CIA by using LDLR-deficient mice. The fact that CIA was more severe in apoE-deficient mice than in LDLR-deficient mice excludes that the LDLR is the mechanism by which ApoE modulates CIA severity. These findings demonstrate that both hypercholesterolemia and ApoE, but not HDL function, regulate the intensity of systemic autoimmune response (Alvarez et al., 2016).

Dietary factors Significant evidence has permitted to identify associations between serum HDL-C and diet, revealing a link between different dietary factors and their potential effects on HDL-mediated atheroprotection (Escola-Gil et al., 2015) Dietary fats The content of the dietary fat plays a key role in the development of CVD and metabolic diseases, such as obesity, atherosclerosis and T2D. Dietary saturated fatty acids (SFA) intake is associated with an increased risk of CVD. Also, a direct relationship between high intake of SFA and HDL-C has been established from several epidemiological studies and one meta-analysis (Knuiman et al., 1987; Mensink et al., 2003; Kotseva et al., 2008). Conversely, polyunsaturated fatty acid (PUFA) intake usually reduces HDL-C, even though they are potentially atheroprotective (Escola-Gil et al., 2015). We initially investigated the effect of a high–SFA, Western-type diet, with or without added cholesterol, on the entire macrophage-to-feces RCT pathway in mice (Escola-Gil et al., 2011). The high–SFA and cholesterol-containing diet caused a significant increase in plasma cholesterol, HDL-C, and liver cholesterol and accelerated the rate of macrophage-to-feces RCT by 3- to 4-fold. These effects were greatly reduced in mice fed the same high-SFA diet without added cholesterol. We also used humanized mice expressing human CETP and, similarly to the findings in wildtype mice, the high–SFA and cholesterol-containing diet induced a significant increase in fecal macrophage-derived [3H]-tracer excretion. The dietary stimulus on RCT was also almost totally absent in CETP transgenic mice fed with the

Diabesity Patients with type 2 diabetes (T2D) show an increased risk for premature CVD and death (Mazzone et al., 2008). Most (~80%) of individuals with T2D are obese, highlighting the pivotal role of increased adiposity as a risk factor (Chadt et al., 2000). Plasma levels of lipids are also frequently altered in both entities (Bays et al., 2013; Tchernof and Despres, 2013), particularly, those of HDL which are often reduced (Kontush and Chapman, 2008). HDL are also dysfunctional in patients with T2D (Kontush and Chapman, 2008). Consistently, cholesterol efflux mediated by HDL from diabetic patients is markedly reduced (Apro et al., 2016). In relation to T2D, compelling experimental evidence suggests that the hepatobiliary trafficking of cholesterol might be impaired in an animal model, the db/db mice, being in part associated with reduced hepatic levels of ABCG5 and G8. We have recently demonstrated that macrophage-to-feces RCT is impaired in db/db mice partly due to defects in the hepatic LXR signalling (Errico et al., 2017). In this case, RCT dynamics may be partly restored by a potent LXR agonist who induces Abcg5/g8. No-

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cardioprotective role in CVD, some epidemiological studies have suggested that phytosterols may have anti-tumorigenic properties (Berges et al., 1995). Some potential mechanisms include alteration in sterol metabolism which may affect cancer cell growth, apoptosis promotion and decreased angiogenesis (Bradford and Awad, 2010). We analyzed the role of phytosterol-supplemented diet in the tumor onset and progression in PyMT mice (Llaverias et al., 2013). A 2% phytosterols supplement caused a significant delay in the development of hyperplasic mammary lesions and a decrease in total tumor burden in PYMT mice fed a high-fat, high-cholesterol diet. Also, Cyclin D1, a tumor biomarker, was shown to be significantly decreased in PyMT transgenic mice fed the phytosterol supplement. However, phytosterols neither affected the availability of cholesterol in the mammary gland nor altered the expression of the main cholesterol transporters. Importantly, phytosterols reduced HDL-peroxidation, thereby enhancing HDL antioxidant capacity. Decreased valules of Laudan GP indicated that this protection might be due to changes in the physicochemical properties of HDL, thereby increasing the packing of the surface lipids and promoting lipoprotein resistance to oxidation. We concluded that dietary phytosterols caused a delay on breast tumor progression in the setting of a typical Western diet and suggest that phytosterols may exert these anticancer effects by preventing oxidative damage. (Llaverias et al., 2013)

SFA diet without cholesterol. When we analyzed the different RCT steps, the serum from mice fed the high–SFA and cholesterol-containing diet had a higher ability to induce macrophage cholesterol efflux. Furthermore, the high–SFA and cholesterol-containing diet-fed mice showed an accelerated excretion of fecal HDL-derived [3H]-cholesterol. The latter was completely blunted when mice were fed the diet without cholesterol. Importantly, only the high– SFA and cholesterol-containing diet increased liver Abcg5/ g8 expression. We also found that this RCT increase was independent of other existing metabolic disarrangements such as obesity or insulin resistant. We concluded that the presence of dietary cholesterol and liver ABCG5/G8 transporters is required for the SFA- and cholesterol-mediated induction of RCT. Since this diet also promoted atherosclerosis, this change in RCT seems to constitute a compensatory mechanism to protect macrophages from cholesterol accumulation (Escola-Gil et al., 2011). Since the high–SFA and cholesterol-containing diet impairs HDL antioxidant function, we investigated whether PUFA could correct the deleterious effects of SFA. We evaluated the effect of replacing dietary SFA by PUFA (mainly the linoleic and α-linolenic acids) on HDL antioxidant potential and the macrophage-specific RCT pathway (Cedo et al., 2015). The high-SFA diet caused a significant increase in serum HDL lipids and upregulated the levels of oxidized (ox) HDL and oxLDL. Replacing dietary SFA with PUFA reverse the effects of SFA on HDL lipid levels and on lipoprotein oxidation. The ability of HDL to reduce LDL oxidation was evaluated by conjugated diene formation of LDL under prooxidant conditions in the presence of HDL. The results demonstrated that HDL from high-SFA and cholesterol-containing diet-fed mice showed an impaired ability to protect against LDL oxidation . Importantly, the PUFA-containing diet prevented the SFA-mediated impairment of HDL antioxidant function. The preferential action of PAF-AH on linoleic acid-containing phospholipids of oxidized lipoproteins might explain the higher ability of PUFA-fed mice HDL to prevent the onset of LDL oxidation. These PUFA-mediated changes in oxidized lipoproteins were not associated with the insulin resistance. In contrast with the critical role of cholesterol on macrophage-to-feces RCT, the amount of dietary PUFA did not affect the rate of RCT pathway in mice (Cedo et al., 2015).

Methionine Methionine-rich foods can affect the plasma levels of homocysteine (Hcy), an intermediate metabolite of the folic acid pathway, causing a hyperhomocysteinemia (HHcy). HHcy induced by methionine can accelerate atherosclerotic progression and consequently has been considered a risk factor for CVD (Dayal and Lentz, 2008). Furthermore, HHcy has been associated to low levels of HDL-C in animal models and in humans (Liao et al., 2007; Vergeer et al., 2010). We determined the effect of HHcy induced by a methionine-rich diet on macrophage-to-feces RCT and on the ability of HDL to protect against LDL oxidation. Methionine-induced HHcy in mice resulted in a significant decrease of HDL-C and apoA-I. The susceptibility of HDL to oxidation was enhanced in HHcy mice. This change was concomitant with an impaired ability of HDL to prevent LDL oxidative modification. Importantly, PON1 and PAF-AH activities, two of the main HDL-associated enzymes with antioxidant activity, were also reduced in HHcy mice. The ability of HDL to efflux cholesterol from macrophages was decreased in HHcy mice; however, dietary methionine did

Phytosterols Dietary phytosterols are able to reduce intestinal cholesterol absorption and consequently decrease the LDL levels on serum (Calpe-Berdiel et al., 2009). Beyond this potential

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HDL function and novel HDL-targeted therapies for preventing atherosclerotic cardiovascular disease

not affect the macrophage-specific RCT measured as the output of macrophage-derived cholesterol into feces (Julve et al., 2013). Overall, our findings indicate that HHcy might exert at least part of its proatherogenic effect by impairing the antioxidant properties of HDL.

peutic approaches and the overexpression and deletion of HDL proteins involved in the major HDL cardioprotective functions. Current data have demonstrated that enhanced macrophage-to-feces RCT or HDL antioxidant functions are inversely correlated with atherosclerosis development. It should be noted that CETP expression did not affect major antiatherogenic functions in mice (Rotllan et al., 2008), which is consistent with the results of 3 synthetic CETP inhibitors that failed to reduce CVD (Barter and Rye, 2012; Lee-Rueckert et al., 2016). However, a new CETP inhibitor, anacetrapib, substantially increased HDL-C and apoA-I, but also pre-β HDL particles and reduced CVD events (Bowman et al., 2017). At present, other HDL-based therapies, such as infusions of apoA-I mimetics or other reconstituted HDL particles for increasing apoA-I levels, appear to be promising tools in preclinical studies. Taken together, the available data indicate that increased HDL-C do not always correlate with enhanced HDL functions and, therefore, should not be considered a biomarker of HDL functionality. In humans, the efficiency of RCT has been evaluated with the surrogate parameter that indicates the ability of HDL to promote the first step of RCT, the macrophage cholesterol efflux capacity. Recent clinical data suggest that this parameter is a strong predictor of CVD in humans (Rohatgi et al., 2014; Saleheen et al., 2015).

Resveratrol Resveratrol, a natural polyphenolic compound, is found in foods such as the skin of grapes, blueberries, raspberries and mulberries. Some cardioprotective properties have been attributed to resveratrol, such as hypolipidemic, antioxidant and anti-inflammatory actions. Some studies in mice indicated that resveratrol may increase HDL-C and, consequently, inhibit atherosclerosis progression (Ramprasath and Jones, 2010). Resveratrol has also been identified as an activator of the NAD+-dependent deacetylase sirtuin1 (SIRT1), although resveratrol has not been proved to activate SIRT 1 directly. Therefore, both mechanisms may involve the modulation of LXR (Pacholec et al., 2010). We investigated the ability of resveratrol and SIRT1 expression to induce LXR-target genes and the macrophage-specific RCT pathway in vivo (Escola-Gil et al., 2013a). In contrast to the effects of the selective LXR-agonist T0901317, no changes in liver cholesterol levels and intestinal cholesterol absorption were observed in mice given resveratrol at different doses. The highest resveratrol dose did not increase HDL-C while the LXR agonist did cause a significant increase. Further, SIRT1 expression in transgenic mice did not affect serum HDL-C and intestinal cholesterol absorption compared with wild-type. However, SIRT1 transgenic mice showed higher non-HDL and liver cholesterol content which has been attributed to elevated liver lipogenesis (Qiang et al., 2011). As expected, mice treated with LXR-agonist presented a higher content of macrophage-derived [3H]-cholesterol in plasma and feces. However, macrophage-to-feces RCT rate was not affected by the treatment of resveratrol. Even though SIRT1 transgenic mice showed differences in the lipoprotein profile, SIRT1 expression did not affect the magnitude of macrophage RCT. Furthermore, the expression of LXR-target genes was unaffected by resveratrol. We concluded that resveratrol and SIRT1 expression did not promote the RCT pathway in vivo (Escola-Gil et al., 2013a).

Acknowledgements This work was partly funded by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, and FEDER “Una manera de hacer Europa”, grants FIS 14-01648 (to F.B.-V.), CP13-00070 (to J.J.), FIS 16-00139 (to J.C.E.-G.), and grants 303/C/2016 (201602-30.31) (to J.J.) and 12/C/2015 (to F.B-V) from La Fundació La Marató TV3. J.J. is recipient of a Miguel Servet Type 1 contract. CIBER de Diabetes y Enfermedades Metabólicas Asociadas is an Instituto de Salud Carlos III Project. Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau is accredited by the Generalitat de Catalunya as Centre de Recerca de Catalunya (CERCA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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HDL function and novel HDL-targeted therapies for preventing atherosclerotic cardiovascular disease

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RESEARCH REVIEWS Institut d’Estudis Catalans, Barcelona, Catalonia

O P E N A ACCE S S

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CONTRIB SCI 13(1):33-36 (2017) doi:10.2436/20.7010.01.258

Bellvitge Biomedical Research Institute: research centre for cancer, neurosciences and translational medicine Gabriel Capellá Bellvitge Biomedical Research Institute (IDIBELL). Gran Via de l’Hospitalet 199, 08908 L’Hospitalet de Llobregat, Barcelona

Resum. L’Institut de Recerca Biomèdica de Bellvitge (IDIBELL), establert l’any 2004 a l’Hospitalet de Llobregat, al sud de Barcelona, té el seu focus en càncer, neurociències i medicina translacional. Els seus socis locals són l’Hospital Universitari de Bellvitge Hospital (HUB) I l’Hospital de Viladecans, que són part de l’Institut Català de la Salut (ICS), l’Institut Català d’Oncologia (ICO), la Universitat de Barcelona (UB) i l’Ajuntament de l’Hospitalet de Llobregat. L’any 2009 es van convertir en un dels cinc primers instituts d’investigació sanitària acreditat per l’Institut de Salut Carlos III. Els investigadors de l’IDIBELL fan recerca bàsica, translacional, clínica i epidemiològica de qualitat que està organitzada en 3 àrees i 9 programes. Aquesta recerca té com a objectiu una aplicació efectiva dels avenços científics en la prevenció, diagnòstic, pronòstic i tractament dels problemes de Salut alhora que promouen la innovació en la recerca sanitària.

Abstract. The Bellvitge Biomedical Research Institute (IDIBELL)  is a biomedical research center for cancer, neurosciences and translational medicine established in 2004 in L’Hospitalet de Llobregat, south of Barcelona. Its local partners are the Bellvitge University Hospital (HUB) and the Viladecans Hospital, both part of the Catalan Institute of Health (ICS), the Catalan Institute of Oncology (ICO), the University of Barcelona (UB) and the town council of L’Hospitalet de Llobregat. In 2009, it became one of the first five Spanish research centers accredited as a  health research institute by the Carlos III Institute of Health. IDIBELL also carries out high-level basic, epidemiological, translational and clinical research though three areas and nine research programs, aiming at an effective translation of scientific advances into the prevention, diagnosis, prognosis and treatment of health problems and promoting innovation in health research.

Keywords: IDIBELL, Cancer, Neuroscience, Translational Medicine, Bellvitge. Correspondence:

Gabriel Capellá gcapella@idibell.cat

The Bellvitge Biomedical Research Institute (IDIBELL) is one of the most dynamic health research institutes in Spain with more than 1200 affiliated researchers in the fields of level basic, epidemiological, translational, clinical and health services research striving to attain significant improvements in human health. Established in 2004, the institute is located in L’Hospitalet de Llobregat, south of Barcelona. In 2009, IDIBELL

e-ISSN: 2013-410X

became one of the first five Spanish research centres accredited as a health research institute by the Institute of Health Carlos III being one of the most productive biomedical research institutions in Spain. IDIBELL encompasses the research activity of the Campus of Bellvitge, including that of the Bellvitge University Hospital and the Viladecans hospital, belonging to the Catalan Institute of Health (ICS), the Catalan Institute of On-

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Bellvitge Biomedical Research

cology (L’Hospitalet), the University of Barcelona (Bellvitge Campus). IDIBELL’s mission is to develop state-of-the-art patient-oriented research that will help into the improvement of the quality of life of citizens. IDIBELL’s vision is to become a global reference in health research while contributing to the development of the Bellvitge Health Area as a hub of excellence capable of attracting research talent in this area. The combination of comprehensive medical care, research capacity and unique economic environment are key assets in this endeavour. The values that inspire our daily task include integrity, commitment, passion, curiosity, transparency and trust. We want that our research has a true impact in society and we also aim at making research closer to society. IDIBELL’s research is structured into three Areas: Cancer, Neurosciences and Translational Medicine, with a total of 9 Research Programs: (Figure 1). Each Research Program is composed of distinct research groups, directed by group leaders, that share a common technical framework or theme. Both single PI (principal investigators) and multiPI groups coexist within the Programs. Research carried out at IDIBELL translates to remarkable scientific productivity; in 2016, IDIBELL’s scientific production was of high quality, as exemplified by 1052 scientific publications indexed in PubMed (56% in Q1 and 22% in the D1), bringing the cumulative impact factor close to 4,291 points and a mean impact factor of 4.8 per article.

Through the years IDIBELL has made a sustained effort in innovation and technology transfer in biomedicine, creating not only value for patients but economic impact as well. In the year 2016: 19 R&D contracts were signed, three active spin-off companies were active, 4 technology transfer agreements were closed and 12 new international patent applications were filed. In the same year, 911 clinical trials were ongoing, 182 having started in 2016). With a total laboratory area of 5000 m2, IwDIBELL not only offers research laboratories but also hosts shared facilities for the support of Clinical Research and for Experimental Research. Clinical Research Support facilities include IDIBELL’s biobank, the clinical trials unit (UICEC) and the biostatistics unit. Experimental Research Support facilities include state-of-the-art cell culture rooms, areas licensed for radioisotope use, unit for the study of molecular interactions, histology, microscopy (including confocal, time-lapse and electronic), flow cytometry, genomics and molecular biology, small animal and zebrafish housing facilities. The core facilities have benefited from the recent alliance with the CMRB (Center for the Regenerative Medicine) now installed in the Hospital i Duran Hospital. In addition to the excellent scientific facilities available, IDIBELL offers researchers a strong network of support departments such as the Research Support Office, the Innovation and Technology Transfer Office, the Communications & Outreach Department, and a Human Resources Depart-

Figure 1. IDIBELL’s research organization

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Capellá

ment. Each department counts on experienced personnel in the relevant area and can be counted on to assist the researcher with the successful implementation of their projects. Of note, IDIBELL has a longstanding track‐record in the participation and successful coordination of European projects. The dedicated European and International Projects Unit in the Research Support Office has helped researchers in managing 41 projects in the FP7 program (3 as coordinators), including 2 ERC (1 Starting and 1 Advanced) as well as 2 IMI projects. So far, IDIBELL has been involved in 8 projects in H2020 (2 as coordinators). IDIBELL also manages 11 projects financed by various prestigious international organisations, including the National Institutes of Health (NIH) and Worldwide Cancer Research (formerly AICR). In addition, IDIBELL manages over 300 national grants. Internationalization has been identified as a strategic objective for IDIBELL. Currently, IDIBELL accommodates international scientists on a regular basis and scientists of several nationalities hold permanent or temporary positions at the institute. IDIBELL endorses the “European Charter for Researchers” and the “Code of Conduct for the Recruitment of Researchers” as key elements to encourage the mobility of researchers and the harmonization of their work conditions across Europe. IDIBELL helps researchers settle in Barcelona and ensure that they can focus on research training and professional development. IDIBELL is in a good position within the Catalan and the Spanish research systems that will help it in facing his main current challenges that include better promoting and executing cutting-edge clinical and translational research, to attract and retain talented researchers, to converge with the Center of Regenerative Medicine of Barcelona and to guarantee its economic sustainability.

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RESEARCH REVIEWS Institut d’Estudis Catalans, Barcelona, Catalonia

O P E N A ACCE S S

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CONTRIB SCI 13(1):37-40 (2017) doi:10.2436/20.7010.01.259

Translational medicine in Catalonia: the case of liver oncology Josep M. Llovet1-3 �Barcelona-Clínic Liver Cancer Group (BCLC), Unitat d’Hepatologia, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic, Universitat de Barcelona, Barcelona. �Mount Sinai Liver Cancer Program, Division of Liver Diseases. Icahn School of Medicine at Mount Sinai, New York, USA. �Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona.

Abstract. Biomedical research has improved during the last decade in Catalonia, which is currently a well-recognized international biomedical scientific hub. The policy of head-hunting and attraction of talent fostered by the Catalan Government through the ICREA Professorship initiative has been successful. In 2006, I was appointed as ICREA Professor and started a research group in Translational research in liver oncology at IDIBAPS-Hospital Clínic in Barcelona. This appointment was possible due to a suitable environment in this institution, both regarding liver and translational medical research. This article summarizes some of the major advancements in translational medicine, liver research and translational hepatic oncology achieved during the last decade.

Resum. La recerca biomèdica ha millorat notablement a Catalunya durant la darrera dècada de tal manera que el nostre país es considera un pol de recerca internacionalment reconegut. Les polítiques endegades pel Govern de la Generalitat amb la creació del programa ICREA per a l’atracció de talent internacional han estat molt exitoses. En el meu cas, vaig tornar l’any 2006 de Nova York per organitzar un grup de recerca translacional en oncologia hepàtica al IDIBAPS-Hospital Clínic de Barcelona. Els avenços del nostre grup han estat possibles atès l’entorn científic que ofereix la institució pel que fa a recerca en malalties hepàtiques i en recerca translacional. El present article analitza alguns dels avenços en recerca translacional, recerca hepàtica i recerca translacional en oncologia hepàtica assolits al nostre país en la darrera dècada.

Keywords: –

Correspondence:

Josep M. Llovet https://www.icrea.cat/Web/ScientificStaff/josep-m-llovet-bayer-387; http://translationalhcc.idibaps.org

Translational medicine in Catalonia Translational medicine has achieved major goals during the last decade in Catalonia. We understand translational medicine as the branch of the biomedical field aimed to

e-ISSN: 2013-410X

translate discoveries from basic research to the clinical setting in order to benefit patients�. Originally, the concept of translational medicine was captured by the motto from bench to bedside, but I rather prefer to describe it as from the patient to the lab and back to the patient�. In most ins-

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Translational medicine in Catalonia

Liver research at IDIBAPS-Hospital Clínic

tances, translational medicine equals to the modern clinical research. In order to put this concept in context in Catalonia, we need to take into account the following facts: a) Advancements produced in translational medicine represent, in my view, the most substantial and critical research produced in biomedical centers in Catalonia, particularly by those centers associated with clinical hospitals, such as IDIBAPS (associated to Hospital Clínic of Barcelona), IDIBELL (associated to Hospital Bellvitge) and VHIR/ VHIO (associated to Hospital Vall d’Hebrón). b) Recent data from the Catalan Government – reported in Science on Sept 29th,2017- between 2007 and 2015 Catalonia has obtained 210 European Research Council (ERC) projects, with a total of 334M€ of funding. Some awarded projects are substantially translational in nature. c) Most of the so called Top 1% cited investigators worldwide working in Catalan institutions are conducting translational research in medicine. d) At least 3 Universities in Barcelona are offering studies in biomedical translational research. Among those, I am directing the Official Master in Translational Medicine, in the Faculty of Medicine, University of Barcelona. To further understand the depth of the achievements in translational medicine in our country, I am summarizing some of the ones that have been generated in my own institution, IDIBAPS-Hospital Clínic. a) Autoimmune encephalitis: Discovery of this new entity -autoimmune encephalitis- and characterization of the NMDA receptor as the target of the immune system. This advancement has re-defined the molecular basis of some mental disorders, thus providing the rationale for a novel therapeutic approach�. b) Chronic lymphocytic leukemia (CLL): Novel classification of CLL and discovery of the landscape of oncogenic mutations as potential targets for therapies�. c) Cancer of unknown origin: This entity is causing thousands of deaths worldwide. A recent study from IDIBELL/IDIBAPS demonstrates that by exploring the methylome of the neoplastic cells it is possible to identify the primary origin of the neoplasm and optimize the treatment�. d) Alzheimer disease: Identification of amyloid-related biomarkers in the asymptomatic phase of Alzheimer’s disease. This discovery paves the path towards primary prevention of this devastating disease�. e) Bipolar disorder: By genome-wide analysis the authors identified a DNA polymorphism able to predict response to lithium for prevention of recurrence of bipolar disorder�.

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The Liver Unit at Hospital Clínic-IDIBAPS is one of the 3-4 international groups with a higher impact in advancing the understanding of the pathogenesis and treatment of liver diseases during the last decades. This Unit was founded and led by Dr Joan Rodés, a giant in the world of Hepatology. He was also founder of the European Association for the Study of the Liver (EASL) and Editor-in-chief of Journal of Hepatology. His outstanding scientific production – h index of 128- was acknowledged with several national and international awards. He had the vision in 1970-80 to organize the research in liver disease according to specific areas (ascites, portal hypertension, hepatitis, liver cancer and transplant), assigning the leadership of each one to different faculties. An overview of the main contributions has been reported elsewhere�·�, and are summarized as follows: a) Staging and treatment of hepatocellular carcinoma (HCC): The proposal of the Barcelona-Clínic system (BCLC) for staging and treatment allocation��·��, has been adopted by European and US clinical practice guidelines of management of HCC. This group has also provided the evidence for establishing chemoembolization as standard of care in intermediate HCC��, and led the international randomized trials supporting the use of sorafenib�� and regorafenib�� in first and second line treatments in advanced cases. b) Pathogenesis and treatment of ascites: Characterization of the mechanisms involved in the development of liver-related complications, such as ascites��. In addition, the authors led several studied supporting the use of albumin infusion to prevent hepato-renal syndrome after large volume paracentesis�� and to improve survival of spontaneous bacterial peritonitis episodes��. c) Treatment of portal hypertension: The group described the pathofisiological mechanisms of portal hypertension and the efficacy of pharmacological�� and non-pharmacological therapeutic approaches��. d) Treatment of hepatitis C infection: The treatment of hepatitis C infection has revolutionized the field during the last 5 years. Nowadays, up to 90% of patients achieve sustained virological responses with the new drug antiviral treatments. The group has contributed to the understanding and management of this infection in the context of chronic liver disease�� and after liver transplantation��. Other important achievements have been reported in the areas of liver transplantation and cholestatic disorders.

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Llovet

Translational research in liver oncology

After analyzing the research achievements described above in the area of translational medicine, liver research and translational liver oncology, it seems obvious to me that successful research is achieved by scientist with a unique fingerprint: a) talented leadership and group, b) adequate environment to facilitate conducting high-end research, c) hard work capacity, this is a constant in all cases, and d) focus on a specific area.

In 2006, and taking advantage of the outstanding research environment in the setting of the Liver Unit and the BCLC group, I created the group of translational research in liver oncology at the IDIBAPS- Hospital Clínic. This occurred after spending 3 years at the Mount Sinai School of Medicine in Nova York, were I created the Liver Cancer Program. Thus, after my return to Europe I retained the position of Director of this program in NYC, which is currently the only NCI recognized liver cancer program in the US. As a result of these two outstanding positions, our group has been able to produce more than 200 research articles in peer-reviewed journals (mean impact factor/article: 18 points). This research has been supported by competitive (including EU and NIH grants) and non-competitive funding of around 20M€. I can proudly say that I have been able to mentor 35 physicians and scientists interested in translational research in liver oncology. As a result of all this effort we have made the following contributions: a) Establishment of the molecular classification of HCC. We proposed a molecular classification of HCC after analyzing the genome of >1000 cases��·��. More recently, we complemented the information by providing the characterization of the immune class of HCC, which is expected to respond to checkpoint inhibitors, and the exclusion class, which is expected to be primary resistant to immune therapies��. b) Characterization of oncogenes and signaling pathways involved in the pathogenesis of HCC. As a result of several studies we defined that the main molecular aberrations occurring in HCC are nowadays non actionable, and only around 30% could be potentially treated with effective drugs��·��. From a more clinical perspective, some early clinical trials are currently ongoing based upon these discoveries. c) Establishment of the molecular characterization of intrahepatic colangiocarcinoma (iCCA) and discovery of novel oncogenic drivers. We proposed a novel classification of iCCA and discovered a novel oncogene resulting from a translocation leading to a fusion protein involving FGFR2��·��. These results have been translated in early clinical trials. d) Involvement in the design of clinical practice guidelines for the management of HCC. I have participated in developing the Clinical Practice Guidelines of management of HCC supported by the EASL (2001, 2012 and 2018) and ESMO (2018).

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Translational medicine in Catalonia

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General Contributions to Science publishes mainly two kinds of articles, specialized reviews and general articles on scientific and technological research (see front cover). Submission of manuscripts Manuscripts submissions are preferably made online, at the journal’s website. Authors are asked to upload the items and provide associated metadata or indexing information to facilitate online searching and for the journal’s own use. Articles can be tracked at any time through the editorial process, and authors can participate in the copy-editing and proofreading of articles accepted for publication by logging in with the username and password provided. Contributions to Science does not charge publication fees. However, processing fees may be requested if the manuscript needs substantial language-editing and copyediting. The amount requested will not depend on the length of the manuscripts but on the time devoted to their editing. In special cases, processing fees can be waived. In no case will the acceptance of manuscripts depend on the authors possibility to pay processing fees. Format of manuscripts All contributions should be typed double-spaced (including references and figure legends) on pages not exceeding 30 cm in height (standard A4 is appropriate), with wide margins, and font (preferable Times New Roman) size not smaller than 12 points. If Greek letters (α, β, χ , et cetera) are required, use the “insert symbol” function to write them. It is the policy of the Journal to publish in English only (authors are recommended to have the manuscript thoroughly checked and corrected before submission). The Editors will warmly appreciate co-operation of authors in preparing papers in a manner that will facilitate the work of editing and publication. For research papers a selfexplanatory abstract without reference to the text—in Catalan language, too—not exceeding 200 words should be provided. It is essential that the author responsible for post publication correspondence (the Corresponding author) should be identified on the manuscript. Manuscript organization The manuscript should begin with the following: 1. Title of the paper containing keywords pertaining to the subject matter. No abbreviations should be used in the title. 2. Author full names (including forenames and initials, if used) and their affiliations. If the publication originates from several institutes the affiliations of all authors should be clearly stated by using superscript numbers after the name and before the institute. 3. Postal address of the author to whom all correspondence (including preprints) is to be sent. Telephone and fax numbers as well as e-mail address should be included to speed up communication. 4. A summary (in English and Catalan languages) not exceeding 200 words. 5. A maximum of 5 keywords, which will be used for compiling the subject index. References References should be cited in the text in square brackets and listed at the end of the paper in alphabetical order. Papers not yet published but accepted by a journal may be cited with the journal’s name followed by “in press”. However, this practice is acceptable only if that author has at least received preprints of the paper. In all other cases, reference must be made to “unpublished data” or “personal communication”. References should be in the following style: Published papers Venugopalan VP, Kuehn A, Hausner M, Springael D, Wilderer PA, Wuertz S (2005) Architecture of a nascent Sphingomonas sp. biofilm under varied hydro­dynamic conditions. Appl Environ Microbiol 71:2677-2686

Accepted unpublished papers Same as above, but “in press” must appear instead of the volume and page numbers Books Miller JH (1972) Experiments in molecular genetics. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Book chapters Lo N, Eggleton P (2011) Termite phylogenetics and co-cladogenesis with symbionts. In: Bignell DE, Yves R, Nathan L (eds) Biology of termites: a modern synthesis, 2nd ed. Springer, Heidelberg, Germany, pp 27-50 Electronic journal articles Citation of articles published in electronic-only journals must provide a DOI (preferable) or URL if they do not have traditional page numbers or electronic article identifiers. For all references, list the first eight authors and then add “et al.” if there are additional authors. Since references that are downloaded from online databases do not always show accents, italics, or special characters, the authors should refer to the PDF files or printed versions of the articles and correct accordingly the submitted manuscript. The journal titles should be abbreviated. Tables Tables should be compiled on separate sheets (one per page table) with a concise, descriptive title and numbered independently of the figures using Arabic numerals in the sequence in which they occur. Every table must be referred to in the text. In the text, the position for a table is to be marked by “Table...” in the middle of an extra line. The caption must explain in detail the contents of the table. As for the table itself, it must be written so that it can be read and understood without reference to the text. References to a table are to be handled in the same way as references to the text (see References). Figures Figures, including photographs should also be submitted on separate pages at the end of the article (new page for each figure). In addition, they must be submitted also in JPG separate files with a resolution of 300 dpi. Figures should be numbered consecutively with Arabic numbers in the order of their appearance. A separate file of fine quality suitable for reproduction is required. Figures should not be larger than the manuscript page. Numbers and symbols inscribed must be large enough to be legible after reduction. If a figure comprises several separate parts, a single composite illustration containing all parts of the figure must be submitted. It is the responsibility of the author(s) to obtain the required permissions to reproduce figures and other material that has been published previously, and the source should be always indicated in a reference. The permission must be obtained from the publishers or where they credited to third parties, from those third parties. Nomenclature, abbreviations and units Internationally standardized nomenclature should be used. Abbreviations and acronyms should be kept to a minimum and spelled out in full at first mention in both the summary and main text. Non-standard abbreviations should be avoided if they appear not more than three times in the text. Only SI units are to be used (SI = Système International d’Unités). If data with non-SI units are to be reported, they should be put in parenthesis behind the corresponding data in SI units. Acknowledgements Acknowledgements to individuals that have contributed to the study and are not named as authors as well as information regarding funding sources should be included in this sections after the main text (before References).

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CONTRIBUTIONS to SCIENCE | VOLUME 13 | Issue 1 | June 2017 Institut d’Estudis Catalans, Barcelona, Catalonia

Gonzàlez Sastre, F.

7

Introduction

Arús, P.

9

Molecular markers for plant genetics and breeding

Canyelles, M.; Cedó, L., Julve, J.; 17 Escolà-Gil J. C.;, Blanco-Vaca, F.

Capellá, G.

Llovet, J. M.

OPEN ACCESS JOURNAL www.cat-science.cat http://revistes.iec.cat/contributions

HDL function and novel HDL-targeted therapies for preventing atherosclerotic cardiovascular disease:From mouse models to human disease

33 Bellvitge Biomedical Research Institute: research centre for cancer, neurosciences and translational medicine 37

Translational medicine in Catalonia: the case of liver oncology

Contributions to Science  

Volume 13 - Issue 1 - June 2017

Contributions to Science  

Volume 13 - Issue 1 - June 2017