Endocannabinoid system and placentaldevelopment: importance in trophoblast cellular turnover and modulation of protein expression

  Mariana Pinto de Almeida Teixeira da Costa Endocannabinoid system and placental development: importance in trophoblast cellular turnover and modulation of protein expression Tese do 3º Ciclo de Estudos Conducente ao Grau de Doutoramento em Ciências Farmacêuticas na especialidade de Bioquímica Trabalho realizado sob a orientação de Professora Doutora Georgina Lopes Correia da Silva (Orientadora) Professora Doutora Natércia Aurora Almeida Teixeira (Co-orientadora) Outubro, 2014 ii  Este trabalho foi realizado no Departamento de Ciências Biológica, Laboratório de Bioquímica da Universidade do Porto e no Grupo de Inflamação e Biologia de Reprodução do Instituto de Biologia Molecular e Celular (IBMC) e com o apoio financeiro da Fundação para a Ciência e Tecnologia (FCT), através da atribuição de uma bolsa de doutoramento (SFRH/BD/70721/2010), no âmbito do POPHQRENTipologia 4.1Formação Avançada, comparticipado pelo Fundo Social Europeu e por fundos nacionais do MEC.  iii  É autorizada a reprodução parcial desta Dissertação/Tese, apenas para efeitos de investigação mediante declaração escrita do interessado, que a tal se compromete; iv  Author’s declaration Under the terms of the “Decreto-lei nº 216/92, de 13 de Outubro”, is hereby declared that the following original articles were prepared in the scope of this dissertation. Under the terms of the referred “Decreto-lei”, the author declares that he afforded a major contribution to the conceptual design and technical execution of the work, interpretation of the results and manuscript preparation of the published articles included in this dissertation. Publications Articles in international peer-reviewed journals M.A. Costa, B.M. Fonseca, N.A. Teixeira, G. Correia-da-Silva. The endocannabinoid anandamide induces apoptosis in cytotrophoblast cells: involvement of both mitochondrial and death receptor pathways. Placenta. 2014. [Epub ahead of print]. DOI: 10.1016/j.placenta.2014.10.011. M.A. Costa, B.M. Fonseca, E. Keating, N.A. Teixeira, G. Correia-da-Silva. Transient receptor potential vanilloid 1 is expressed in human cytotrophoblasts: induction of cell apoptosis and impairment of syncytialization. The International Journal of Biochemistry & Cell Biology. 2014.[Epub ahead of print]. DOI: 10.1016/j.biocel.2014.10.008. Costa MA, Keating E, Fonseca B, Teixeira N, Correia-da-Silva G. 2-Arachidonoylglycerol impairs human cytotrophoblast cells syncytialization: Influence of endocannabinoid signalling in placental development. Molecular and Cellular Endocrinology. 2014; [Epub ahead of print]. doi: 10.1016/j.mce.2014.09.005. Costa MA, Fonseca B, Keating E, Teixeira N, Correia-da-Silva G. 2-arachidonoylglycerol in cytotrophoblasts: Metabolic enzymes expression and apoptosis in BeWo cells. Reproduction. 2014;147:301-11. doi: 10.1530/REP-13-0563. v  Abstracts in international peer-reviewed journals M. A. Costa, B. M. Fonseca, N. A. Teixeira and G. Correia-da-Silva. 2arachidonoyglycerol (2-AG) and placental development: role of endocannabinoids in cytotrophoblast cells turnover (2012). FEBS Journal 279 (Suppl. 1); pp63; P01-37. Abstracts published in congress books Costa, Mariana A., Keating E., Fonseca B. M., Teixeira N. A., Correia-da-Silva G. The Endocannabinoid System in cytotrophoblast differentiation: 2-arachidonoylglycerol impairs in vitro syncytialization of human cytotrophoblast cells. XLIV Reunião Anual Sociedade Portuguesa de Farmacologia. 5th-8th February 2014. Coimbra (Portugal). (Oral communication). A. C. Gomes, MA Costa, BM Fonseca, NA Teixeira and G Correia-da-Silva. BeWo cells differentiation: Syncytialisation induction and anandamide effects in cell viability. 7th Meeting of Young Researchers of University of Porto, Porto 12-14 Fevereiro, 2014. M.A. Costa, B.M. Fonseca, V. Guimarães, N.A. Teixeira, G. Correia-da-Silva. The endocannabinoids anandamide and 2-arachidonoylglycerol in placenta: impact in viability and proliferation of trophoblast cells. 3rd I3S Scientific Retreat, Porto, 10-11 May, 2012. V Guimarães, MA Costa, BM Fonseca, NA Teixeira and G Correia-da-Silva. The endocannabinoid 2-arachidonoylglycerol (2-AG) in pregnancy: effects in viability of cytotrophoblast cells 5th Meeting of Young Researchers of University of Porto, Porto 22-24 Fevereiro, 2012. M.A. Costa, B.M. Fonseca, V. Guimarães, N. A. Teixeira and G. Correia-da-Silva. The endocannabinoids anandamide and 2-arachidonoylglycerol in placenta: impact in viability and proliferation of trophoblast cells. V SPB Clinical Biochemistry WorkshopTranslational Molecular Biochemistry, Coimbra (Portugal), 27 Janeiro, 2012. vii  Agradecimentos Às Professoras Doutoras Georgina Correia da Silva e Natércia Teixeira, minhas orientadoras, agradeço os conhecimentos transmitidos, a disponibilidade, o incentivo, o rigor científico e, sobretudo, a oportunidade de realizar este trabalho. À Professora Georgina pela paciência, optimismo e constante motivação que me proporcionou desde o núcleo de investigação da FFUP, quando eu era ainda aluna de Mestrado Integrado, aumentando e cimentando assim o meu gosto pela ciência. Aos docentes e restantes elementos do Laboratório de Bioquímica da FFUP, nomeadamente ao Bruno, pela paciência e pelas discussões científicas. À Ana Paula, D. Casimira e ao Daniel pela disponibilidade e eficiência. À Professora Doutora Elisa Keating, pela sua simpatia e disponibilidade para me transmitir os seus conhecimentos sobre culturas primárias. A todos os estudantes, docentes e funcionários do Serviço de Bioquímica da FMUP, por me terem acolhido e integrado. Ao Departamento de Obstetrícia e Ginecologia do Hospital de S. João e à Dra. Alexandrina Mendes do Centro Materno-Infantil do Norte pela colaboração neste trabalho. À Fundação para a Ciência e Tecnologia (FCT), pela bolsa de doutoramento que me foi atribuída (SFRH/BD/70721/2010). Por último, à minha família, especialmente aos meus pais, pelo incentivo e apoio incondicional. ix  Abstract The development of human placenta is a highly dynamic and tightly regulated process that involves proliferation, differentiation and apoptosis of specialized epithelial cells, the trophoblasts. Cytotrophoblasts (CTs) are able to proliferate and to differentiate into the non-proliferative multinucleated syncytiotrophoblast (ST) or develop an invasive phenotype, forming the extravillous trophoblasts, which remodel the uterine tissues and blood vessels. The syncytiotrophoblast is in direct contact with the maternal blood and is responsible for the mother-foetus gas and nutrient exchange and for the placental endocrine function. Anomalies during trophoblast proliferation, differentiation and apoptosis are related to pregnancy-associated complications, such as preeclampsia, intrauterine growth restriction or spontaneous miscarriages. In the last years, the relevance of the endocannabinoid system (ECS) in different physiological and pathological processes has been recognized. It is constituted by the cannabinoid receptors (CB1 and CB2), their endogenous ligands (endocannabinoidseCBs) and the biosynthetic and degradation enzymes of the endocannabinoids. The two major endocannabinoids are anandamide (AEA) and 2-arachidonoylglycerol (2-AG). They participate in neuroprotection, apoptosis, nociception and reproductive events. In fact, the endocannabinoid signalling is critical for fertility, implantation and decidualization. However, its importance during placental development remains unclear, though CB receptors and AEA metabolic enzymes have already been identified in this organ. In this way, one of the major aims of this work was to search for the presence of other key members of ECS, namely 2-AG metabolic enzymes (DAGL-α, MAGL) and the transient receptor potential vanniloid 1 (TRPV1), an AEA target, in primary human cytotrophoblasts syncytiotrophoblasts and in the cytotrophoblast cell model BeWo. It was demonstrated that these proteins are expressed in these cells. Then, the role of endocannabinoids in cytotrophoblast proliferation, differentiation and apoptosis and the underlying signalling pathways were investigated. AEA and 2-AG induced apoptosis and oxidative stress in BeWo cells, through a CB receptor-dependent mechanism. TRPV1 activation by AEA or its agonist capsaicin (CPS) also triggered apoptosis in human CTs. In addition, 2-AG impaired the biochemical and morphological differentiation of CTs into STs by the activation of CB receptors and these effects may be related to the 2-AG-induced decrease in cyclic AMP (cAMP) levels and protein kinase A (PKA) PKA phosphorylation. CPS also inhibited CT biochemical differentiation through a TRPV1-dependent mechanism, showing the importance of this receptor in cytotrophoblast differentiation. No significant AEA xvi  List of abbreviations ∆ψm mitochondrial membrane potential 12-,15-HETE-EA 12-,15-hydroxyeicosatetraenoic acid ethanolamide 12-,15-HETE-GE 12-,15-hydroxyeicosatetraenoic acid glycerol ester 12-,15-HPETE-EA 12-,15-hydroperoxyeicosatetraenoic acid ethanolamide 12-,15-HPETE-GE 12-,15-hydroperoxyeicosatetraenoic acid glycerol ester 2-AG 2-arachidonoylglycerol 2-AGE 2-arachidonoyl glyceryl ether 3-β-HSD 3-β-hydroxysteroid dehydrogenase AA arachidonic acid ABHD12 α/β-hydrolase 12 ABHD4 α/β-hydrolase 4 ABHD6 α/β-hydrolase 6 AC adenylyl cyclase AEA N-arachidonoylethanolamine AIBP anandamide intracellular binding proteins ATF-4 activating transcription factor 4 ATP adenosine triphosphate cAMP cyclic AMP CB cannabinoid CB1 cannabinoid receptor 1 CB2 cannabinoid receptor 2 CBD cannabidiol c-FLIP cellular FLICE-like inhibitory protein (c-FLIP) COX-2 cyclooxigenase 2 CPS capsaicin CRE cAMP response element CREB cAMP response element binding protein CSF colony-stimulating factor CT cytotrophoblast CYP450 cytochrome P450 DAG 1,2-diacylglycerol DAGL diacylglycerol lipase eCB endocannabinoid ECS endocannabinoid system EGF epidermal growth factor EMT endocannabinoid membrane transporter xvii  enEVT endovascular trophoblast Epac exchange protein directly activated by cAMP ER endoplasmic reticulum ERK extracellular signal-regulated kinase ESC endometrial stromal cells EVT extravillous trophoblast FAAH fatty acid amide hydrolase FABP fatty acid binding protein FAK focal adhesion kinase FAN factor associated with neutral sphingomyelinase activation FLAT FAAH-like anandamide transporter FSH follicle stimulating hormone GASP1 G-protein receptor-associated sorting protein GC giant trophoblast cell GCM-1 glial cells missing 1 GDE1 glycerophosphodiesterase 1 GM-CSF granulocyte-macrophage colony-stimulating factor GnRH gonadotrophin-releasing hormone GPCR G-protein-coupled receptor GP-NAE glycerophospho-N-acylethanolamine GPR55 G-protein-coupled receptor 55 hCG human chorionic gonadotropin HERVs human endogenous retroviral genes HLA human leucocyte antigen hPL human placental lactogen HSP heat shock protein ICIS intracytoplasmic sperm injection ICM inner cell mass iEVT interstitial trophoblast IGF-1 insulin growth factor 1 IP3 inositol triphosphate IUGR intrauterine growth restriction IVF in vitro fertilization JNK c-jun N-terminal kinase LH luteinizing hormone LIF leukemia inhibitory factor LOX lipoxygenase LPA lysophosphatidic acid MAG monoacylglycerol xviii  MAGL monoacylglycerol lipase MAPK mitogen-activated protein kinase MFSD2a major facilitator superfamily domain-containing protein 2a MMP matrix metalloproteinase NAAA N-acylethanolamine-hydrolyzing acid amidase NADA N-arachidonoyldopamine NAEs N-acylethanolamines NAGly N-arachidonoylglycine NAPE N-arachidonoyl-phosphatidylethanolamine NAPE-PLD N-acylphosphatidylethanolamine-specific phospholipase D NAT N-acyltransferase PA phosphatidic acid pALP placental alkaline phosphatase PARP-1 poly (ADP-ribose) polymerase 1 PC phosphatidylcholine PE phosphatidylethanolamine PG-EA prostaglandin-ethanolamide PG-GE prostaglandin-glyceryl ester PI phosphatidylinositol PI3K phosphoinositide 3-kinase PKA protein kinase A PKB protein kinase B PLA1 phospholipase A1 PLA2 phospholipase A2 PLC phospholipase C PP13 placental protein 13 PPAR peroxisome proliferator-activated receptor PTPN22 protein tyrosine phosphatase non-receptor type 22 PTX pertussis toxin RNS reactive species of nitrogen ROS reactive species of oxygen RXR retinoid X receptor SHIP1 SH2 domain-containing inositolphosphatase SM sphingomyelin SMase sphingomyelinase ST syncytiotrophoblast STAT3 signal transducer and activator of transcription 3 TASK-1 TWIK-related acid-sensitive K+ channels 1 tBid truncated Bid xix  TGF transforming growth factor THC ∆9-tetrahydrocannabinol TIMP tissue inhibitor of metalloproteinase TNF tumour necrosis factor TRAIL TNF-related apoptosis-inducing ligand TRIB-3 telomere repeat binding factor 3 TRPV1 transient receptor potential vanilloid 1 uPA urokinase plasminogen activator VGCC voltage-gated calcium channels VSMC vascular smooth muscle cells XIAP X-linked inhibitor of apoptosis 1  PART I Introduction 3  1. Development and functions of human placenta A new human life begins with the fertilisation of an oocyte by a spermatozoa, giving rise to the first cell of the new being, the zygote. Then, the zygote goes through successive mitosis originating the morula, which undergoes compaction forming the blastocyst. The blastocyst has an outer epithelial cell layer, the trophectoderm, which will form the placenta and extra-embryonic tissues; and an inner cell mass (ICM), which will give rise to the embryo. The blastocyst implantation occurs after 5-7 days of fertilization. For a successful implantation, it is required that the endometrium becomes receptive to the blastocyst, after differentiation of endometrial stromal cells (ESCs) into decidual cells. In humans, the decidualization occurs every menstrual cycle, in the late secretory phase, to prepare the endometrium for the implantation. If fertilisation does not occur, decidua is expelled during the menstruation and the endometrium is regenerated. If fertilisation occurs, the decidua is maintained and the endometrium undergoes further changes that include vascular and glandular remodelling and the appearance of the uterine immune cell populations. The formation of placenta begins with the differentiation of the trophectoderm in different trophoblasts, the specialized epithelial cells of placenta. In the prelacunar stage, trophoblast cells proliferate and differentiate into the multinucleated syncytiotrophoblast (ST), which has an invasive phenotype in this stage, allowing the blastocyst penetration into the decidua. The remaining mononuclear cells are the cytotrophoblasts (CT) and act as stem cells, permitting the growing of ST layer. In the lacunar stage, fluid-filled spaces appear in the syncytiotrophoblast and merge, forming the lacunae. The lacunae are surrounded by the trabeculae (a ST mass), which will be important for the villous trees formation. At this stage, CTs invade the trabeculae and reach the maternal tissues, giving rise to the extravillous trophoblasts (EVTs). In the villous stage, the trabeculae begin to branch and contain a core filled with CT, forming the primary villi. Then, the extraembryonic mesodermal cells go along with the CTs and invade the trabeculae, though not reaching the maternal side, giving rise to the secondary villi. The mesodermal cells differentiate into haematopoietic cells and, then, blood vessels independent on embryo vascular system emerge, originating the tertiary villi [1]. At microscopic level, placenta is then constituted by villous trees that keep emerging from the continuous branching of trabeculae. Some of them contact with the basal plate (region facing the endometrium) and contain the trophoblastic cell columns (anchoring villi), while other end freely in the intervillous space (floating villi) [1]. This placental 4  microarquitecture allows an intimate contact between mother and foetus, since the maternal blood is in direct contact with trophoblast foetal cells, due to the deep invasion of maternal tissues. This type of placentation is called haemochorial [2]. Macroscopically, the human term placenta is a discoid organ with about 22 cm of diameter but there is a considerable interindividual variability of its anatomy. The basal plate is divided in lobes/cotyledons, which contain the chorionic villi that arise from the foetal side of placenta (the chorionic plate) and are irrigated by foetal blood vessels [1]. The placental development is a dynamic and highly regulated process and anomalies in this gestational event will impair the important functions of human placenta, such as gas and nutrient exchanges and embryo protection [3], compromising the success of pregnancy outcome. The main functions of this organ are summarized in Figure 1. Figure 1. Main functions of human placenta. hCGhuman chorionic gonadotropin; hPLhuman placental lactogen; PP13placental protein 13. 1.1 The trophoblast In placental environment, there are several cell types that contribute to placental structure and function, including trophoblasts, decidual cells, mesenchymal cells, Hofbauer cells (placental macrophages) and endothelial cells [1]. The trophoblasts are the main placental cells. In human placenta, there are four types of trophoblasts: cytotrophoblast, syncytiotrophoblast, extravillous trophoblasts, and giant trophoblast cells. They have different morphologies, properties and functions, which turns the placenta into a unique organ. Figure 2 represents the mother-foetal interface, with the structure of human chorionic villi and the trophoblast cell types. Nutrient supply: aminoacids, glucose and lipids, H2O, vitamins, inorganic ions Gas exchanges Removal of waste products Metabolism Protection: immunoprotection, barrier against xenobiotics Endocrine funtion: hCG, oestrogens, progesterone, hPL, leptin, PP13, Embryo support 5  Figure 2. Representation of the mother-foetal interface. CTs are able to proliferate and fuse to originate a multinucleated layer, the ST. In the anchoring villi, CT form a cytotrophoblast cell column. Then, CTs differentiate into invasive trophoblasts, which will remodel the maternal decidua (iEVTs) and blood vessels (enEVTs), enlarging the spiral arteries caliber and consequently diminishing the resistance to the blood flow that irrigates the foetus. In the myometrium, to limit their invasion into maternal tissues, iEVTs fuse, giving rise to the multinucleated GCs. CTcytotrophoblast; DC-decidual cell; ECendothelial cell; enEVTendovascular extravillous trophoblast; GCgiant trophoblast cell; iEVTinterstitial extravillous trophoblast; MCmyometrial cell; ST-syncytiotrophoblast. The villous cytotrophoblasts are the first trophoblast cell type to differentiate from the trophectoderm. They are mononuclear cells that proliferate, differentiate into other trophoblast cell types and die by apoptosis. Their proliferation is essential for the placental development and maintenance. CT proliferation is regulated by oxygen and growth factors and is higher in the first trimester, when the oxygen tension is lower, decreasing in the following trimesters [4]. Cytotrophoblast differentiation occurs by two pathways: the villous and the extravillous pathways (Figure 3). In the villous pathway, CTs proliferate, aggregate, fuse and undergo biochemical differentiation to originate the syncytiotrophoblast; this process is called syncytialization. In the extravillous pathway, CTs proliferate and acquire invasion properties, which allow the invasion of uterine arteries (endovascular trophoblasts; enEVTs) and endometrium (interstitial trophoblasts; iEVTs) [3, 5, 6]. 12  1.2.3 Oestrogens Placental oestrogens are steroids that consist in four different hormones: oestrone, oestradiol, oestriol and oestretol. On the contrary to other steroidogenic organs, placenta is not able to convert pregnenolone and progesterone into androgens, since 17αhydroxylase/17, 20-lyase is absence in this organ. In this way, placenta uses circulating androgens that essentially come from foetal adrenal glands (but also from the mother), and the enzyme CYP450 aromatase expressed in the syncytiotrophoblast converts them into oestrogens [97]. Aromatase activity is modulated by cAMP levels [100]. Oestradiol promotes implantation, vasodilatation and increases uterine contractions in labour. However, oestradiol actions in the trophoblasts remain unclear. Moreover, oestradiol enhances syncytialization, induces leptin expression and may, indirectly, modulate EVTs function [98, 101]. The oestriol is the most abundant oestrogen in the urine. It is considered a weak oestrogen, though it is important for increasing uteroplacental blood flow and is one of the hormones tested for screening foetal anomalies [97]. 1.2.4 Leptin and other adipokines Adipokines are adipocyte-derived signalling proteins that, in the last years, have emerged as important regulators of gestational events, namely in placental development. Leptin is the most well-known adipokine in pregnancy. Serum levels of leptin are increased in pregnant women, peaking in late 2nd and early 3rd trimester. The main source of leptin is the syncytiotrophoblast and leptin receptors are also expressed in this multinuclear layer. This hormone has pleotropic effects in pregnancy, such as in implantation, angiogenesis, embryo development, stimulation of hCG secretion, CT proliferation and induction of MMP-2 and MMP-9 synthesis and, consequently, EVTs invasion. Leptin triggers JAK/STAT, ERK1/2 and Phosphoinositide 3-kinase (PI3K) pathways [102]. Its expression is regulated by other pregnancy hormones like hCG, hPL, insulin, progesterone, oestrogens and by the second messenger cAMP. In fact, cAMP induces the expression of leptin, in a mechanism that also involves a crosstalk between PKA and ERK1/2 pathways [103]. Additionally, hCG enhances the production of leptin through a mechanism that involves an interplay between cAMP and p38 [104] and also by the Epac/cAMP pathway [105]. Adiponectin is another adipokine whose importance in placentation has been recognized. It exerts several tissue-specific effects, partially depending on the several 13  isoforms found in the circulation: trimmers, hexamers and high molecular weight multimers. Its serum levels are increased in early pregnancy and then decline in the second trimester [106]. Adiponectin diminishes the endocrine function of term syncytiotrophoblast, since it decreases hCG, progesterone and hPL secretion [107]. On the other hand, it enhances syncytialization in first trimester [33] and has antiproliferative effects on trophoblastic cell lines, BeWo and JEG-3 [108]. Moreover, adiponectin stimulates the migration and invasion of first trimester EVTs, through the upregulation of MMP-2 and -9 and downregulation of TIMP-2 [109].This cytokine also inhibits insulin signalling and insulin-stimulated amino acid transport [110] through the activation of PPAR-α and ceramide synthesis [111]. Resistin serum levels are increased in the 3rd trimester of gestation. This adipokine is expressed in EVTs and increases MMP-2 whereas decreases TIMP-1 and -2, promoting cell invasion. Resistin also stimulates angiogenesis [106]. 14  2. The Endocannabinoid System The consume of Cannabis sativa either for medicinal or recreational purposes began a thousand years ago in central Asia. The medicinal properties of this plant were first explored in China, India, Egypt, Syria, Persia and Tibet, where it was used for the treatment of malaria, constipation, rheumatism, pain, absentmindedness, menstrual fatigue, sleep disorders [112-114]. However, the first cannabinoids were only identified in the 60’s: the Cannabidiol (CBD) [115] and the most psychoactive constituent of cannabis, ∆9tetrahydrocannabinol (THC) [116]. After the identification of THC, an intense research about its mechanism of action began. The strong hydrophobicity of this molecule suggested that its actions resulted from non-specific interactions with cell membrane [117]. However, the synthesis of THC enantiomers and its synthetic analogues, as well as the discovery that THC was enantioselective indicated that THC effects were receptor-dependent. In 1988, the first cannabinoid receptor (CB1) was identified in rat brain by Devane et. al. [118] and in 1992, the first endogenous ligand of this receptor, the N-arachidonoylethanolamine or Anandamide (AEA), was discovered in porcine brain [119]. Then, in 1993, the second cannabinoid receptor CB2 was identified in human promyelocytic leukemia cells (HL-60 cells) [120]. These discoveries and the identification of other endogenous ligands for CB1 and CB2 contributed to unveil the existence of the Endocannabinoid System (ECS). Nowadays, the members that constitute this system are the cannabinoid receptors (CB1 and CB2), their endogenous ligands (endocannabinoids – eCBs), the enzymes involved in synthesis and degradation of these ligands and the eCBs putative membranar transporter (EMT). In Figure 4, the members of ECS are represented, as well as their role in the system. In the last years, the ECS has been studied in several scientific fields and it has emerged as an important intervener in different physiological and pathophysiological mechanisms. Actually, drugs targeting the members of ECS have been explored as therapeutic alternatives for the treatment of pathological conditions such as pain, cancer, obesity, inflammation and neurodegenerative disorders [121]. The drugs that target ECS are divided in several families such as direct agonists or antagonists of CB receptors and inhibitors of eCBs biosynthesis, degradation or reuptake [122]. Cannabinoid agonists, like THC or the synthetic cannabinoid nabilone are legally used in some countries to treat nausea and emesis in cancer patients undergoing chemotherapy and to induce appetites in AIDS patients. These compounds have also proved efficacy in the management of 15  interferon and ribavirin-induced anorexia, nausea and weight loss in chronic hepatitis C patients. In Portugal, a spray containing THC and cannabidiol (CBD), Sativex®, is commercialized since 2012 for the treatment of spasticity in multiple sclerosis patients [123]. The selective antagonist of CB1 receptor, Rimonabant (Acomplia®) was a promisor anti-obesity drug and was commercialized in several countries. However, Acomplia® was discontinued due to its psychiatric side effects (anxiety and depression) [124]. Figure 4. Biochemistry of the endocannabinoid system (ECS). Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are synthetized from membrane precursors. AEA is synthesised by the enzymes N-acyltransacylase (NAT) and N-acylphosphatidoylethanolaminePhospholipase D (NAPE-PLD), whereas the synthesis of 2-AG is catalysed by Phospholipase C (PLC) and Diacylglicerol lipase (DAGL). Both eCBs are released to the extracellular environment by diffusion or selective endocannabinoid membrane transporter (EMT), where they activate G.protein-coupled cannabinoid receptors (CBR). Then, they are rapidly internalized also by diffusion /EMT and are degraded. AEA is mainly hydrolysed by fatty acid amid hydrolase (FAAH), releasing ethanolamine (EA) and arachidonic acid (AA) or is oxidized by Cyclooxygenase 2 (COX-2), to prostaglandin-ethanolamides (PG-EA). 2-AG is converted to glycerol and AA mainly by Monoacylglycerol lipase (MAGL) or is oxidized by COX-2 to prostaglandin-glyceryl esters (PG-GE). In addition, AEA is capable of activating the cation channel transient receptor potential vanilloid 1 (TRPV1) receptor in the intracellular side and both AEA and 2-AG are able to bind to the nuclear receptors peroxisome proliferator-activated receptor (PPAR), modulating the transcription of their target genes. 16  2.1 Endocannabinoids By definition, endocannabinoids are endogenous lipid molecules that are capable of binding to and functionally activate cannabinoid receptors, exerting similar cellular effects to the most psychoactive constituent of Cannabis sativa, the THC. As abovementioned, the first eCB N-arachidonoylethanolamide (Anandamide, AEA) was identified in 1992 in porcine brain [119]. In the following years, other eCBs have also been identified: 2-arachidonoylglycerol (2-AG) [125, 126], 2-arachidonoyl glyceryl ether (2-AGE) [127], O-arachidonoylethanolamine (virodhamine) [128], Narachidonoyldopamine (NADA) [129], N-arachidonoylglycine (NAGly)[130] and Oleamide [131]. Chemically, these molecules are amides, esters or ethers of long-chain polyunsaturated fatty acids and are structurally different from THC but share critical pharmacophores [132, 133]. They are synthesised in cytoplasm through multiple biosynthetic pathways and released into the extracellular environment, where they bind and activate CB receptors. After exerting their action, eCBs are internalized and degraded [134]. The molecular structure of THC and eCBs are represented in Figure 5. All eCBs have different affinities for cannabinoid receptors and exhibit differences in their pharmacological profile comparing with THC and synthetic cannabinoids. Some eCBs are able to activate other receptors like the Transient Receptor Potential Vanilloid Type-1 (TRPV1) and PPAR-α and γ [135]. Anandamide and 2-AG are the best characterized members of the main eCBs families, the N-acylethanolamides (NAE) and monoacylglycerols (MAG), respectively. Since the pharmacology and metabolism of these compounds are the most well-known, they are considered as ‘major’ endocannabinoids. Besides eCBs, there are other endogenous molecules, “endocannabinoid-like”, which are able to modulate the endocannabinoid signalling, although they are devoid of activity on CB receptors. These molecules are denominated eCB-like compounds and share the hydrolysing enzymes with the eCBs, exacerbating their effects by an ‘entourage’ effect, due to the competitive inhibition of these enzymes [136]. 2.1.1 Anandamide (AEA) N-arachidonoylethanolamine or Anandamide is the best-characterized eCB. The name Anandamide comes from the Sanskrit word ananda, which means ‘internal bliss’ [137]. As the other NAEs, this lipid is an endogenous eicosanoid derivative, which is synthesized from phospholipids of cell membrane and arachidonic acid, linked by an amide bound. Its 17  main biosynthetic enzyme is N-acyl-phosphatidylethanolamine (NAPE-PLD), whereas the main hydrolysing enzyme is Fatty Acid Amide Hydrolase (FAAH) [134]. Figure 5. Chemical structures of the psychoactive phytocannabinoid THC and of the endogenous cannabinoids. It is known that AEA is very important in numerous physiologic processes like modulation of different stages of reproduction [138-141], nociception [142, 143], muscle relaxation [144, 145], inflammation [146, 147], stimulation of appetite [148, 149], cell differentiation [150-152] and apoptosis [138, 153-155]. For a long time, researchers thought that AEA and other eCBs were synthetized ‘on demand’. However, recent data ∆ 9 -Tetrahydrocanabinol (THC) Anandamide (AEA) 2-Arachidonoylglicerol (2-AG) 2-Arachidonoylglicerol Ether (2-AGE) Virodhamine N-arachidonoyldopamine (NADA) N-arachidonoylglycine (NAGly) Oleamide (ODA) 18  demonstrated that AEA can be stored inside the cell in adiposomes [156]. Moreover, AEA can interact with soluble carrier proteins facilitating its trafficking [157, 158]. Anandamide acts as partial agonist of both cannabinoid receptors, which is unusual for an endogenous ligand. Like THC, AEA has lower efficacy and affinity for CB2 than for CB1 [159]. Besides cannabinoid receptors, AEA can also activate TRPV1, a non-selective cation channel [160] and so, this eCB is also considered an endovanilloid. This receptor can also be responsible for some AEA cellular effects, like vasodilation and apoptosis [161]. AEA can also activate the nuclear receptors PPAR-α [162] and PPAR-γ [163]. Interestingly, AEA is able to downregulate the levels of the other major eCB, 2-AG, through activation of TRPV1 in postsynaptic neurons, resulting in an inhibition of 2-AG synthesis by Diacylglycerol Lipase α (DAGL-α) [164]. In this way, AEA is capable of acting inside the cell through the activation of TRPV1 and PPARs and exerting an autocrine and/or paracrine effect on CB receptors when it is released from the cell [165]. AEA in the extracellular fluid can reach different targets with the help of protein carriers like serum albumin and lipid-binding proteins, such as lipocalins [166], acting as an endocrine messenger [165]. 2.1.2 2-arachidonoylglycerol (2-AG) 2-arachidonoylglycerol was an already known endogenous metabolite when, in 1995, Mechoulam et al. identified it in canine gut and described its intrinsic activity on CB1 and CB2 [125]. Sugiura et al. also found that this eCB binds to the cannabinoid receptors in rat brain synaptosomes [126]. There are emergent evidences that 2-AG is an important messenger in multiple physiological processes such as neuroprotection [167, 168], inflammation [169-171], nociception [172, 173], reproductive events [174, 175], cell differentiation [176, 177] and apoptosis [175, 178]. Similarly to AEA, 2-AG is synthesised in the cytoplasm through different pathways and the most relevant is the one involving the enzyme diacylglycerol lipase (DAGL). The major 2-AG hydrolysing enzyme is monoacylglycerol lipase (MAGL) [134]. 2-AG binds to CB1 and CB2 receptors with higher efficacy than AEA but the affinity for CB2 is slightly lower than for CB1 [159]. Unlike AEA, 2-AG acts as full agonist in various systems and seems that its structure is strictly recognized by the cannabinoid receptors, which leads some authors to conclude that 2-AG is the true natural ligand for the CB receptors [179]. It has been referred that the concentration of 2-AG in the brain is between 170 [180] to 800 [126] times higher than AEA. Also, in mouse uterus and in rat decidual 19  cells, 2-AG levels are, respectively, 200and 150-fold higher than those of AEA [174, 175]. In contrast with AEA, 2-AG seemed to be a very weak activator of TRPV1 [160], though recent evidences support that this eCB is also a ligand of this receptor [181, 182]. 2-arachidonoylglycerol, as AEA, activates PPAR-γ [163]. 2.2 Receptors for the endocannabinoids Cannabinoid receptors, CB1 and CB2, are members of the superfamily of G proteincoupled receptors (GPCR), which have a seven-domain transmembrane structure. Their ligands bind to the extracellular domain, activating different cellular pathways. Initially, it was thought that THC effects resulted from its interaction with cellular membranes, which would stimulate or inhibit membrane-associated enzymes and alter the physical state of ion channels [117, 183]. However, the finding that THC induced an inhibition of AC and a decrease in the levels of cAMP in neuroblastoma cells constituted the first evidence of the existence of cannabinoid receptors [184, 185]. These inhibitory effects were blocked by pertussis toxin (PTX), suggesting the involvement of Gi/0 proteins [186]. In 1988, a study using a radiolabelled and enantiomerically pure analogue of THC allowed the identification of specific binding sites in the brain, the CB1 receptors [118]. In 1990, these receptors were cloned from rat brain [187]. Later, in 1993, the discovery of CB2 demonstrated that it only shares 44% of amino acid sequence homology with CB1 [120]. While CB1 receptor is highly conserved across the human, rat and mouse species, CB2 protein structure is divergent, which may have implications for future pharmacological studies involving CB2 [188]. Initially, it was thought that CB1 was only localized in brain and that CB2 was restricted to immune system, however, their distribution is more ubiquitous. Besides brain, CB1 is peripherally expressed in several organs, including spleen, tonsils, bladder, small intestine, sympathetic nerve terminals, smooth muscle cells and reproductive tissues [134]. CB2 receptor is mainly expressed in periphery especially in lymphoid organs and immune cells but it was also identified in brain tissues [189]. Other difference between the two cannabinoid receptors is the physical association of CB1 with lipid rafts in several cell types, since these membrane microdomains are important for the modulation of its activity [190-193]. In fact, it was reported that the disruption of lipid rafts with methyl-β-cyclodextrin enhances the AEA-induced effects through CB1 by interfering with mechanisms responsible for attenuation or termination of CB1 signalling [192]. On the other hand, cholesterol enrichment of plasma membrane reduced the CB1 receptor-dependent signal transduction, due to the increased rigidity 20  [194]. Also, lipid rafts are important for CB1 internalization by endocytosis [190, 195, 196], which leads to degradation in lysosomes or recycling, regulating CB1 levels in the cell membrane. On the contrary to CB1, CB2 binding and signalling is not dependent on the microarchitecture of the plasma membrane [197-199]. This may result from the putative cholesterol recognition amino acid sequence present in CB1 [200] or due to the posttranslational palmitoylation of this receptor [201], that seem to be important for CB1 interaction with lipid rafts. Nevertheless, it was also described that lipid rafts disruption reverses AEA-induced cell death in different cell types, through receptor-independent mechanisms [138, 202, 203]. In the last years, evidences supporting the existence of other putative cannabinoid receptors have been emerged. The orphan GPCR, GPR55, is the best candidate to be considered the third cannabinoid receptor (CB3), since it seems to be activated by cannabinoid receptors agonists, including AEA and 2-AG [204]. Besides cannabinoid receptors, it has been reported that endocannabinoids interact with other type of receptors and modulate the activity of ion channels. Transient Receptor Potential Vanilloid (TRPV1) is a non-selective cation channel belonging to the sixtransmembrane-domain Transient Receptor Potential (TRP) channels and it was identified in 1997 by Caterina et al. [205]. This receptor is activated by noxious heat (> 42 ºC), low pH (<6) and by several endogenous and exogenous molecules and its best known agonist is the capsaicin (CPS), an hot chilli pepper component [206]. TRPV1 is expressed in several organs and tissues among the body and participates in different physiological events. In fact, this receptor is mainly involved in temperature sensing [207] and nociception [205, 208, 209], but is also associated with other cellular processes such as apoptosis [153, 210, 211], muscle contraction [212-214], cell differentiation [215-217], autophagy [218, 219] and inflammation [208, 220, 221]. It is known that AEA and 2-AG may also target the nuclear receptors PPARs involved in the regulation of metabolism and energy homeostasis, cell differentiation, immune/inflammatory responses and reproductive events. [162, 163, 222-224]. 2.3 Metabolism of Endocannabinoids The endocannabinoids, mainly AEA and 2-AG, are involved in several important physiological mechanisms, so their biosynthesis and degradation must be tightly regulated to maintain the homeostasis of endocannabinoid signalling. 21  2.3.1 Biosynthesis of Anandamide and other N-acylethanolamines N-acylethanolamines like AEA can be synthetized through multiple pathways (Figure 6). They are originated from plasma membrane phospholipids [225] and their major biosynthetic pathway, transacylation–phosphodiesterase pathway, is catalysed by the enzymes N-acyltransacylase (NAT) and N-acylphosphatidoylethanolamine-Phospholipase D (NAPE-PLD). NAT is a calcium-dependent enzyme localized in cellular membranes, which transfers a fatty acid chain from sn-1 position of 1, 2-diacylglycerophospholipid (e.g. phosphatidylcholine –PC) to the amine group of phosphatidylethanolamine (PE). From this reaction results the precursor of NAE, N-acylphosphatidylethanolamine (NAPE), and a lysophospholipid [226]. NAPEs are biological constituents of plasma membrane and their levels are increased in inflammation and tissue degeneration [226]. The second step of NAE formation is catalysed by NAPE-PLD, a calcium-dependent and highly conserved enzyme belonging to the zinc metallo-β-lactamase family. This enzyme is also localized in biological membranes and converts Narachidonoylphosphatidylethanolamine (NArPE) into AEA and other NAPEs into their correspondent NAE, releasing phosphatidic acid [227]. Although the transacylation-phosphodiesterase pathway is considered the major source of AEA, NAPE-PLD knockout mice revealed the existence of alternative biosynthetic pathways [228]. One pathway involves the double O-deacylation of NAPEs by the serine hydrolase α/β-hydrolase 4 (ABHD4), which is expressed in several organs. ABHD4 hydrolyses NAPE to lyso-NAPE and then lyso-NAPE to glycerophospho-Nacylethanolamine (GP-NAEs) [229]. GP-NAE is finally converted into NAEs by the metaldependent GP-NAE phosphodiesterase, glycerophosphodiesterase 1 (GDE1) [230]. In addition, secreted Phospholipase A2 (sPLA2) is expressed in several rat tissues especially in stomach and is also able to convert NAPE to lyso-NAPE, which is then converted to AEA by lyso-Phospholipase D (lyso-PLD) [231]. Another pathway of AEA synthesis involves the synthesis of phospho-Narachidonoylethanolamine (pAEA) by a not yet identified Phospholipase C (PLC)–like enzyme. pAEA is subsequently dephosphorylated by a protein tyrosine phosphatase (PTPN22), originating AEA. Inositol 5’-phosphatase SHIP1 also contributes to the synthesis of AEA from pAEA [232]. 28  In addition, AEA uptake can also occur via a protein-carrier-mediated caveolae-related endocytosis. This process proposes that AEA binds a carrier protein located within the caveolae, a caveolae-derived vesicle (clathrin-independent) is formed and, subsequently, AEA endocytosis occurs; this process is rapid, saturable and energy independent [282]. In this way, the eCBs uptake is still poorly understood and more information about this process is required. Figure 10 resumes the proposed pathways of AEA transport across cell membrane and a model of intracellular trafficking of AEA. Figure 10. The proposed models for AEA uptake and intracellular traffic. After activating cannabinoid receptors in extracellular environment (CBR), AEA can passively diffuse across cellular membrane due to a concentration gradient (represented at left, in dark blue). AEA may be transported into the cells via a protein carrier-mediated caveolae-related endocytosis (represented in centre, in blue). Endocannabinoid Membrane Transporter (EMT) may facilitate the internalization of AEA (represented at right, in light blue). AEA may be sequestered inside the cell by binding to AEA Intracellular Binding Proteins (AIBPs) or stored in adiposomes, which may also interact with AIBPs. Moreover, FAAH-like AEA transporter (FLAT) may also participate in AEA uptake. All these proteins may distribute AEA throughout the cytoplasm and cell organelles, facilitating its degradation by fatty acid amide hydrolase (FAAH). Inside the cell, AEA can also activate the transient receptor potential vanilloid 1 (TRPV1) or nuclear receptors [134]. The majority of these models were suggested after studies of AEA transport however, there are evidences that suggest that AEA and 2-AG are accumulated in cells via common mechanisms. Moreover, 2-AG inhibits AEA cellular uptake indicating a competitive nature of the transport of these two eCBs [271]. 29  2.4 Endocannabinoid Signalling Cannabinoid receptors are expressed in several cells and tissues in the organism. Along with their agonists, CB receptors are crucial for the transduction of endocannabinoid signalling, which modulates multiple cellular pathways in different physiological and pathophysiological mechanisms. The signalling pathways affected after CB receptors activation are summarized in Figure 11. CB receptors are generally coupled to Gi/0 proteins, triggering several cellular mechanisms, like AC inhibition, MAPK activation, activation of inward rectifying K+ (Kir) channels or inhibition of voltage-gated Ca2+ channels [283, 284]. The coupling of CB1/2Gi/0 was demonstrated by the impairment of these effects after treatment with PTX, a specific inhibitor of Gi/0 proteins [186, 283, 285]. Nevertheless, under conditions of PTX treatment, CB1 but not CB2 may interact with Gs [286] and Gq/11 [287] proteins leading to different cellular effects. The inhibition of some AC isoforms by Gi/0 proteins-coupled to CB receptors results in a decrease of cAMP levels, a second messenger that stimulates the activity of Protein Kinase A (PKA). Consequently, reduced PKA activity impacts important cellular signalling events including voltage-dependent current flow at A-type K+ channels [288] and focal adhesion kinase (FAK) phosphorylation [289]. Besides Gi/0, CB1 may also be coupled to Gs proteins [290]. Recently, it has been described that CB receptors activation induces the phosphorylation of cAMP response element binding protein (CREB) [291, 292]. MAPK pathway regulates cellular functions such as proliferation, differentiation and apoptosis. CB1 and CB2 activation increases the phosphorylation of p38 [293-297], ERK 1/2 [298-302] and c-Jun N-Terminal Kinases (JNK) [296, 303, 304]. Moreover, CB receptors activation enhances [305, 306] or inhibits the Phosphoinositide 3-kinase (PI3K)/ Protein Kinase B (PKB or Akt) [307]. Cannabinoid agonists regulate ionic channels through activation of CB1. In fact, they inhibit voltage-gated calcium channels (VGCC) of most types including P/Q, N and L-type channels by a mechanism mediated by Gi/0 proteins [308-310]. T-type Ca2+ channels are also inhibited by AEA through direct binding [311]. Furthermore, CB1 activation promotes the stimulation of Kir channels, also in a Gi/0-dependent way [312]. TWIK-related acidsensitive K+ channels 1 (TASK-1), which are responsible for setting membrane potential, are inhibited by AEA and other CB agonists [313]. CB receptors activation may also increase intracellular Ca2+ concentrations through different pathways that aims the release of Ca2+ from IP3-sensitive stores [314-316]. 30  Figure 11. Cannabinoid signalling pathways resulting from the activation of CB receptors coupled to G protein. The inhibition of AC induces a decrease in cAMP levels, leading to a decrease in PKA activity. CB1 also mediates the stimulation of intracellular kinases such as ERK, JNK, p38, FAK and PI3K/AKT, which are involved in several intracellular mechanisms. Ionic channels can also be regulated by cannabinoid signalling receptors: Ca 2+ channels inhibited whereas K + channels are induced after the activation of CB1. Moreover, the intracellular levels of Ca 2+ are increased. The binding of the adaptor protein FAN to CB1 induces ceramide accumulation through the stimulation of SM hydrolysis by SMase. Also, CB1 can stimulate de novo synthesis of ceramide by SPT, from FA and Ser. AC-Adenylyl Cyclase; ERKExtracellular signal-regulated kinase; FAfatty acid; FAKfocal adhesion kinase; FANfactor associated with neutral sphingomyelinase activation; JNKcJun N-Terminal Kinases; PI3K-Phosphoinositide 3-kinase; PKA-Protein Kinase A; Serserine; SMsphingomyelin; SMase-Sphingomyelinase; SPTserinoylpalmitoyltransferase. The adaptor protein factor associated with neutral sphingomyelinase activation (FAN) is also able to interact with CB1, mediating its interaction with sphingomyelinase (SMase), the enzyme that hydrolysis sphingosine into ceramide. In this way, CB1 triggers an acute generation of ceramide, which is related to the regulation of metabolic functions. Nevertheless, activation of CB1 also induces a long term ceramide accumulation due to the stimulation of its de novo synthesis by serine palmitoyltransferase [317]. Ceramide mediates CB1-induced apoptosis in several cell types [297, 318-321]. Besides CB1, CB2 also stimulates ceramide synthesis, inducing apoptosis [155, 321]. Recently, some proteins that specifically interact with CB1 were discovered. CB1 receptor interacting proteins, CRIP1a and CRIP1b, bind to CB1 C-terminal but, till now, only CRP1a effects on modulation of CB1 activity were recognized [322]. G protein- 31  coupled receptor associated sorting protein 1 (GASP1) interacts with CB1 receptor, inducing its downregulation and regulating its post-endocytic targeting to lysosome [323]. β-arrestins are also involved in desensitization of CB1 receptor, since they bind to agonistoccupied CB1, preventing the signal transduction and initiating the endocytosis of CB1arrestin complex [324]. Since some of these proteins reside within or depend on lipid rafts, it is possible that the disruption of these structures could negatively influence CB1 desensitization, exacerbating CB1 signalling [325]. 2.5 Endocannabinoid system in cell death Cell death is a cellular event that is crucial for the homeostasis of tissues and organs, while unbalanced cell death rates are associated with several pathological conditions such as cancer, neurodegenerative diseases and pregnancy-related complications. The ECS has emerged as an important intervener in cell death processes like apoptosis, necrosis or autophagy. Apoptosis is a programmed cell death mechanism by which cells die without damaging the neighbouring cells. This is an ATP-dependent process and occurs mainly by two major apoptotic pathways, the mitochondrial and the death receptor pathways. Cells undergoing apoptosis own morphological characteristic features such as chromatin condensation and fragmentation and membrane blebbing. The pro-apoptotic effects of AEA have been described in several cell types, through CB receptor-dependent and independent mechanisms, triggering different downstream signalling pathways. In fact, after binding to CB1 or CB2, AEA induces apoptosis by activation of caspase-9, loss of ∆ψm, oxidative stress and ceramide accumulation [297], as well as, cytochrome c release [155, 192, 326] or intracellular Ca2+ levels increase [327]. Also, AEA-induced apoptosis may require the phosphorylation of p38, ERK1/2 or JNK [297, 326-329], which may occur directly after CB1 activation or ceramide accumulation. Besides AEA, 2-AG can also induce apoptosis in some cell types, such as decidual cells [175] and hepatic stellate cells [178], through the activation of mitochondrial pathway. CB1 and CB2 activation may also trigger an apoptotic mechanism mediated by the endoplasmic reticulum (ER) stress. In fact, in human pancreatic tumor cells, a CB2 receptor-dependent accumulation of de novo synthetized ceramide upregulates the stress-regulated protein p8, the ER stress-related genes activating transcription factor 4 (ATF-4) and telomere repeat binding factor 3 (TRIB-3) [330]. 32  Most of the abovementioned apoptotic mechanisms involve the activation of the mitochondrial pathway. Nevertheless, eCBs may also trigger the death receptor pathway. In fact, in cholangiocarcinoma cells, AEA causes apoptosis through a CB receptorindependent mechanism, by accumulation of ceramide and recruitment of Fas and FasL into lipid rafts [331] and recently, these effects were attributed to the activation of GPR55 [332]. In Chang liver cells, AEA mediates cell death by activation of both extrinsic and intrinsic pathways, with upregulation of FasL, Bim and Bax [333]. The CB2 agonist JWH015 and THC also induce apoptosis in immune cells, through a crosstalk between extrinsic and intrinsic pathways with activation of caspases-3,-8 and -9 and loss of ∆ψm [334, 335]. In addition, in chondrocytes, Gomez et al. reported that AEA induces apoptosis through a CB receptor-independent mechanism that also involves an interplay between intrinsic and extrinsic pathways [154]. In addition, the activation of TRPV1 by AEA triggers the mitochondrial pathway in human neuroblastoma and lymphoma cells [336] and, in human endothelial cells, AEA-induced activation of TRPV1 leads to apoptosis by a mechanism dependent on p38 and JNK phosphorylation [337]. A role for COX-2 oxygenated metabolites of AEA, the prostamides, in cell apoptosis has recently emerged. In keratinocytes, AEA mediates apoptosis after being metabolized to J-series prostaglandins, which generate oxidative stress [250]. Moreover, the AEAinduced cell death in colon cancer cells is also dependent on COX-2 but independent on oxidative stress [249]. Recently, in melanoma cells, it was reported that AEA proapoptotic effects are, at least partially, mediated by COX-2 and also LOX metabolites [338]. The eCBs have also been associated with necrosis. In fact, AEA and 2-AG induce necrosis in rat decidual cells [138, 175]. AEA induces necrosis in hepatic stellate cells, by increasing intracellular Ca2+ levels and oxidative stress [339] and through the downregulation of the prosurvival pathway PI3K/Akt and induction of inflammatory cytokines production [202]. Autophagy is a cellular event pivotal for cell homeostasis that is involved in both cell survival and death. It consists in enclosing cellular components into autophagosomes (double-membrane vesicle), which will fuse with lysosomes to be degraded and recycled. Recently, new data have reported the involvement of the endocannabinoid signalling in autophagy-related apoptosis. In fact, THC stimulates de novo synthesis of ceramide via CB1 in glioma cells, initiating an ER stress response, which induces autophagy via TRIB3dependent inhibition of Akt/mTORC1 (mammalian target of rapamycin complex 1) axis, a repressor of this cellular event. Moreover, it was reported that autophagy is required for the cannabinoid antitumoral action, since it was crucial for the activation of the 33  mitochondrial pro-apoptotic pathway [340]. In hepatocellular carcinoma, THC and CB2 agonist JWH-015 induce an autophagic process that is also important for cell apoptosis. Here, autophagy is induced either by activation of adenosine monophosphate-activated kinase (AMPK) or by ceramide accumulation. The latter leads to TRIB3 activation, inhibiting Akt/mTORC axis and activating PPAR-γ, which leads to autophagy and then to cell apoptosis [341, 342]. In addition to the aforementioned cell death mechanisms, in some types of mantle lymphoma cells, the CB1 agonist WIN55-212,2 induces paraptosis, a non-apoptotic programmed cell death mechanism characterized by the presence of cytoplasmic vacuoles of ER origin, which differ from the autophagic vacuoles, since they do not fuse with lysosomes [343]. In summary, the participation of endocannabinoid signalling in different cell death processes seems to be cell specific, since it may trigger different cell death pathways and interfere with several cellular signalling mechanisms. 34  3. Endocannabinoids in reproduction The hazards of Cannabis sativa in reproductive function are known for years. Its major psychoactive component, THC, induces a decline in pituitary hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin, and also in sex hormones, progesterone, oestrogens, and androgens, having a negative outcome in fertility. THC also inhibits ovulation, crosses placenta and can accumulate in maternal milk [344-346]. The consumption of cannabis during gestation is implicated in deficient foetal growth, low birth weight, preterm labour and long-term neurobehavioral disturbances [347, 348]. With the discovery and identification of CB receptors and their endogenous compounds that are capable of mimicking the THC effects, an interest in the study of their implications in physiologic and pathophysiologic mechanisms has emerged. Also, CB receptors are present in female and male reproductive organs and tissues like ovaries, uterus, placenta, testis, prostate, sperm and embryo. Thus, ECS has been a target for the study of male and female fertility and of different stages of pregnancy like preimplantation, implantation, decidualization, placentation, foetal development and labour. Figure 12 summarizes eCBs effects on these reproductive events. 3.1 Endocannabinoid system in female reproductive tract and fertility The presence of eCBs in several female reproductive tissues, organs and fluids suggests a role for this system in physiological reproductive processes. AEA is present in mid-cycle oviductal fluid, follicular fluid, amniotic fluid, milk and in human seminal plasma, suggesting a role for AEA in modulation of multiple physiological and pathophysiological processes, such as follicular maturation, ovulation, placental and foetal development and lactation. Human ovaries express both cannabinoid receptors in cortex and medulla and CB2 expression is generally higher. FAAH and NAPE-PLD are expressed only in secondary and tertiary follicles, corpus luteum and corpus albicans, which enable the AEA synthesis in the ovaries [349]. Peralta et al. recently demonstrated that human oocytes express CB1 and CB2 and that their localisation change during the various stages of meiotic resumption [350]. During the menstrual cycle, plasmatic AEA levels suffer alterations, peaking in the early follicular phase [351]. Moreover, the plasmatic peak of AEA is achieved at the ovulation and, then, AEA levels decrease in early luteal phase [352]. 35  During menstrual cycle, it is also verified a correlation between AEA levels and oestradiol, FSH and LH suggesting that these hormones may have a role in regulation of this eCB [352]. In the periovulatory phase, while progesterone synthesis is supressed, the lowest levels of this hormone coincide with lowest levels of FAAH. Also, both molecules reach maximal values at luteal phase. [353]. Together, these data suggest a role for endocannabinoid system in folliculogenesis, follicle and oocyte maturation and ovulation. Figure 12. Schematic representation of endocannabinoids (eCBs) effects throughout pregnancy and the negative impact of an abnormal endocannabinoid signalling. Decidual cells result from the differentiation of endometrial stromal cells, in order to turn the uterus receptive to the implanting embryo. Trophoblast cells proliferate, differentiate, invade maternal tissues and undergo apoptosis, allowing the placental development. Emerging data indicate that eCBs are part of the complex network of hormones, cytokines and other molecules that regulate the reproductive events, namely implantation and decidualization. Furthermore, it is suggested that an abnormal endocannabinoid signalling (yellow boxes) may be involved in the pathophysiology of pregnancy-associated complications, such as intrauterine growth restriction, preeclampsia and spontaneous abortion [354]. The members of ECS are also expressed in human uterus, during menstrual cycle. CB1 and CB2 receptors are widely distributed in uterine tissues. CB1 is expressed with higher intensity in endometrial glands than in stromal cells, whereas CB2 receptor immunoreactivity is minimal at the beginning of a new cycle, increasing till its maximal expression in late-proliferative phase. Moreover, it was verified an increase of FAAH and 36  a decrease in NAPE-PLD expression during menstrual cycle in endometrium till midsecretory phase [355]. Recently, Scotchie et al. corroborated some of these findings reporting that FAAH expression was enhanced in secretory phase but they also reported augmented NAPE-PLD expression. Moreover, they described an increased expression of MAGL and COX-2 in this phase, supporting that low levels of 2-AG may also be required during the implantation period [356]. The eCBs seem also to have a role in the regulation of human myometrial function. In fact, in the human myometrial smooth muscle cell line ULTR, AEA activates ERK1/2 pathway in a CB1-dependent mechanism and inhibits AC, inducing cell viability loss [357]. Recent data, in human myometrium, reported that NAPE-PLD, FAAH and CB1 are expressed but TRPV1 and CB2 are absent in this tissue [358]. In endometrial stromal cells, the non-hydrolysable AEA analogue methananadamide induces cell migration via CB1-dependent activation of PI3K/Akt and ERK1/2 pathways [359]. Moreover, methanandamide enhances ESC proliferation and, longer exposures, induces apoptosis [360]. During human ovulatory cycle, there are oscillations in the levels of AEA and FAAH in peripheral lymphocytes. The expression of NAPE-PLD, EMT and CB receptors remain constant during the cycle [353]. The highest FAAH levels and the concomitant lowest AEA levels are found in the luteal phase, suggesting that these levels may be important for a successful implantation [353, 361]. The evidences of interplay of sex steroids, cytokines and eCBs result essentially from the modulation of FAAH, the major controlling enzyme of AEA, by oestrogen, progesterone, leptin and Th cytokines, indicating a role for eCBs-hormone-cytokines in the regulation of human fertility [362]. Maccarrone et al. demonstrated that progesterone stimulates FAAH activity in human lymphocytes [363]. In addition, profertility Th2 cytokines (IL4, IL10) stimulate FAAH activity whereas the antifertility Th1 cytokines (IL2, IL12 and interferon γ) have the opposite effect [363]. It was already verified that the activation of FAAH by progesterone results from an increase of nuclear levels of transcription factor Ikaros, which binds to FAAH promoter, inducing FAAH gene expression [364]. The adipokine leptin also induces activation of FAAH in human T lymphocytes through the activation of signal transducer and activator of transcription 3 (STAT3), which upregulates a cAMP response element (CRE)-like site in FAAH promoter [365]. Thus, leptin and progesterone seem to exert a synergic effect in FAAH upregulation and consequently in the modulation of AEA levels. Furthermore, leptin and progesterone upregulate FAAH activity in human lymphoma cell line U937 [366]. Moreover, since they 37  decrease AEA levels, leptin and progesterone have a protective role in immune cells through the inhibition of AEA pro-apoptotic effects [367]. In addition, in the uterus of leptin knockout mice, AEA and 2-AG levels are elevated comparatively with wild-type mice, due to the decreased FAAH and MAGL activities and higher activity of DAGL [368]. Together, the data suggest that leptin and progesterone may interfere with the endocannabinoid signalling, reinforcing that abnormal levels of these hormones may be implicated in infertility. Figure 13 schematises the network in human lymphocytes, where FAAH has a central role. Figure 13. Role of Endocannabinoid system in hormone-cytokine network. After its biosynthesis, AEA is released in the intracellular space, where it activates CB1 and, among other actions, inhibits LIF release. AEA is transported into the cell via the putative EMT and is hydrolysed by FAAH to AA and ethanolamine. FAAH is upregulated by a synergic process mediated by progesterone and leptin. Progesterone binds to its intracellular receptor and upregulates the FAAH promotor through the transcription factor Ikaros (Ik). Leptin activates its membrane receptor, leading to the activation of STAT3, which will bind to CRE-like site, activating the transcription of FAAH gene. Moreover, FAAH activation by progesterone is enhanced by Th2 cytokines and inhibited by Th1 cytokines. AAarachidonic acid; AEAanandamide; CREcAMP response element; EMTendocannabinoid membrane transporter; FAAHfatty acid amide hydrolase; IkIkaros; LIF-leukemia inhibitory factor; STAT3signal transducer and activator of transcription 3. 45  PART II Experimental section              47  Manuscript I  2-arachidonoylglycerol in cytotrophoblasts: Metabolic enzymes expression and apoptosis in BeWo cells. MA Costa, BM Fonseca B, E Keating, NA Teixeira, G. Correia-da-Silva Reprinted from Reproduction, 2014; 147:301-11. doi: 10.1530/REP-13-0563. Copyright © 2014 Biosicentifica 61       Manuscript II  The endocannabinoid anandamide induces apoptosis in cytotrophoblast cells: involvement of both mitochondrial and death receptor pathways. MA Costa, BM Fonseca, NA Teixeira, G. Correia-da-Silva Placenta, 2014.DOI:10.1016/j.placenta.2014.10.011 Copyright © 2014 Elsevier Ltd.    The endocannabinoid anandamide induces apoptosis in cytotrophoblast cells: involvement of both mitochondrial and death receptor pathways M.A. Costa1,2, B. M. Fonseca1,2, N. A. Teixeira1,2, G. Correia-da-Silva1,2 1Departamento de Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal 2 Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal Key words: Anandamide; apoptosis; cytotrophoblasts; endocannabinoid signalling; placenta Correspondence to: Georgina Correia da Silva Faculdade de Farmácia da Universidade do Porto Departamento de Ciências BiológicasLaboratório de Bioquímica Rua de Jorge Viterbo Ferreira n.º 228, 4050-313 PORTO – PORTUGAL Telephone: +351 220 428 500 Fax: +351 226 093 390 E-mail: [email protected] ABSTRACT Introduction: A balanced proliferation, apoptosis and differentiation in trophoblast cells of the human placenta is crucial for a proper placental development. Alteration in trophoblast apoptosis and differentiation are associated with gestational-related complications, such as preeclampsia, intrauterine growth restriction or miscarriages. The endocannabinoids (eCBs) have been recognized as new interveners in pregnancy events such as implantation and decidualization. However, their importance in placentation is poorly understood. We hypothesise that these novel lipid mediators may intervene in cytotrophoblast apoptosis and, concomitantly, have a role during placental development. Methods: primary human cytotrophoblasts (hCTs) and the human trophoblast-like choriocarcinoma cell line BeWo cells were exposed to Anandamide (AEA). It was investigated the cellular pathways involved in cell death, by the assessment of cell morphology, caspases activity, mitochondrial membrane potential (∆ψm), reactive oxygen/nitrogen species (ROS/RNS) and western blot of cleaved Poly (ADP-ribose) polymerase 1 (PARP-1), truncated Bid (t-Bid) and IκB-α. Results: AEA decreased hCTs viability and induced morphological features of apoptosis (chromatin condensation and fragmentation), caspase-3/7 activation and PARP-1 cleavage. In BeWo, AEA also increased the activities of caspase3/7 and 9, induced loss in ∆ψm and production of ROS/RNS. These effects were reversed by either CB1 or CB2 antagonists, whereas the increase in caspase 3/7 activity was only reversed with CB2 blockage. AEA-treated cells showed increased caspase-8 activation and formation of t-Bid, suggesting the interplay between intrinsic and extrinsic apoptotic pathways. AEA also increased IκB-α expression, a NF-κB regulatory protein. Conclusion: Our results highlight the importance of eCBs in cytotrophoblast cell apoptosis and indicate that a crosstalk between intrinsic and extrinsic apoptotic pathways is involved in AEA-induced effects. 1   1. Introduction The placenta is a specialized organ with vital functions, such as nutrient and gas exchange, immunomodulation and protein biosynthesis. The main placental cells, the trophoblasts, are subdivided in different cell types: cytotrophoblasts, syncytiotrophoblast and extravillous trophoblasts (EVTs). Cytotrophoblasts are mononuclear cells that proliferate, fuse and differentiate into other types of trophoblasts. The syncytiotrophoblast is a multinucleated layer that produces several hormones and proteins and is in direct contact with maternal blood, allowing the mother-foetus communication. The EVTs have invasive properties and are involved in the uterine blood vessels remodelling [1]. A coordinated proliferation, differentiation and apoptosis of trophoblasts is required for a proper placental development, and any disturbance in these processes is associated with gestational complications [2-7]. The Endocannabinoid System (ECS) has emerged as a key modulator of multiple physiological and pathophysiological processes, including reproductive events like implantation and decidualization. This system comprises the two cannabinoid receptors CB1 and CB2, their endogenous ligands (endocannabinoidseCBs), the enzymes involved in eCBs biosynthesis and degradation and a putative membrane transporter (See [8] for review). Anandamide (AEA) or Narachidonoylethanolamine was identified in 1992 in pig brain [9] and is currently the best studied eCB. It is synthesised from membrane phospholipids in a two-step reaction catalysed by the enzymes N-acyltransacylase and Nacylphosphatidylethanolamine-phospholipase D (NAPE-PLD). After exerting its effect, it is hydrolysed inside the cell, mainly by Fatty Acid Amide Hydrolase (FAAH), an inner cellular membrane enzyme. This eCB induces apoptosis in several cell types by activating different receptors and cell signalling pathways. In fact, it was described that AEA-induced apoptosis involve the activation of its main targets, the CB receptors, triggering several downstream pathways, including activation of mitogenactivated protein kinases (MAPKs), increase of ceramide levels or intracellular Ca2+ and generation of oxidative stress [10-14]. The importance of cannabinoid signalling homeostasis was only been recently recognized. In fact, a proper AEA tone is required for embryo transport along the mouse oviduct [15] and low levels of this eCB are required in implantation sites and are mainly regulated locally by the enzyme FAAH (see [16] for review). Nevertheless, information about the role of AEA during the period of placental development is lacking. It was reported that CB receptors and AEA main metabolic enzymes are expressed in first trimester and term placentas [17-20]. Also, in preeclamptic placentas, NAPEPLD expression was increased whereas FAAH was decreased in comparison to normal placentas, suggesting a function for eCBs in the pathophysiology of preeclampsia [21]. It was also described that AEA decreases the viability of BeWo cells, via CB2 receptor [20]. Moreover, we have recently demonstrated that the other major eCB, 2-arachidonoylglycerol (2AG), has the ability to induce apoptosis in BeWo cells through a CB receptorand mitochondrialdependent mechanism [22]. Here, we investigate the impact of AEA on cytotrophoblasts viability and explore the cellular mechanisms triggered by this endocannabinoid. We hypothesise that this major endocannabinoid may integrate the network of hormones, cytokines and other molecules that regulate cytotrophoblast proliferation and apoptosis. 2. Materials and Methods All chemicals were from Sigma-Aldrich Co. (St. Louis, MO, USA), except: Anandamide, AM251 and AM630 (Tocris Bioscience, Bristol, UK); 3, 3′-dihexyloxacarbocyanine iodide (DiOC6) (Gibco/Invitrogen Corporation, Carlsbad, CA, USA); Z-VAD-fmk (BD PharMingen, San Diego, CA); CytoTox 96 nonradioactive cytotoxicity assay kit, CaspaseGlo® (Promega, Madison, WI, USA); 3Hthymidine (Amersham, Aylesbury, UK); percoll (GE Healthcare, Buckinghamshire, UK); and WesternBrightTM ECL HRP substrate (Advansta, Menlo Park, USA). 2   2.1 Isolation and culture of human cytotrophoblasts All the procedures were performed in accordance with the Ethical Committee of Hospital S. João, Porto (authorization nº 23713). For each assay, cytotrophoblasts were isolated from five different term placentas from clinically normal pregnancies, according to a modification of the Kliman’s protocol, as previously described [23]. Briefly, decidual tissue was removed, villous tissue was collected from at least 10 different regions homogeneously distributed in the whole placenta, and the major blood vessels were discarded by fine dissection. Then, the tissue was digested in a trypsin and DNAse-containing solution and the obtained cells were separated in a discontinuous percoll gradient, at 1200 g. The cytotrophoblasts were collected and incubated at 37 °C and 95% air/ 5% CO2 humidified atmosphere, in DMEM/F12 medium supplemented with 10% (v/v) FBS and antibiotic–antimycotic solution (100 U/ml penicillin G, 100 μg/ml streptomycin, 0.25 μg/ml amphotericine B). To evaluate the purity of the cytotrophoblast cultures, cells were fixed with a 4% paraformaldehyde solution and immunostained with anti-cytokeratin-7 and antivimentin antibodies. Around 95% of the cells were cytokeratin-7 positive, which corresponds to the epithelial hCT cells. For the experiments, cells were plated in 6and 96-well plates or 8well chamber slides, at densities 4.5 x 105, 1.5 x 105 or 5 x 105 cells/well, respectively. 2.2 BeWo cell culture conditions The human choriocarcinoma cell line BeWo (ATCC, Manassas, VA, USA) was incubated at 37 °C and 95% air/ 5% CO2 humidified atmosphere, in DMEM/F12 medium supplemented with 10% (v/v) FBS and an antibiotic–antimycotic solution. For the experiments, cells were used between 78 and 90 passages and seeded in 96 or 6-well plates and 8well chamber slides, at densities 1 x 104, 2 x 105 and 3 x 104 cells/well, respectively. At least five independent experiments performed in triplicate for each assay. For the investigation of cellular pathways triggered by AEA, BeWo cells were pre-treated for 30 min with CB1 and CB2 antagonists, AM251 or AM630 (1 μM), respectively. AEA, AM251 and AM630 were dissolved in ethanol. Equimolar concentrations of the vehicle did not induce any effects on all the parameters (data not shown). 2.3 Cell viability and cytotoxicity assays The hCTs and BeWo cells were plated in 96well plates and, after adhesion, incubated in DMEM/F12 medium with 1% FBS and 1% antibiotic solution, in the absence or presence of AEA (1-25 µM) for 12 and 24 h (hCTs) or for 24, 48 and 72 h (BeWo cells). The yellow tetrazole MTT (0.5 mg/ml final concentration) was added and cells were incubated for 2 h 30 min, at 37 ºC. The resultant purple formazan was extracted by a solution Dimethylsulfoxide (DMSO):isopropanol (3:1) and quantified by spectrophotometry, at 540 nm, in a Multiscan Ascent microplate reader. The activity of the cytoplasmic enzyme lactate dehydrogenase (LDH) released into the culture medium was assessed by the CytoTox 96 nonradioactive cytotoxicity assay kit, according to the manufacturer’s instructions. 2.4 Morphological studies The alterations in cell morphology induced by AEA (15 μM) treatment of hCTs for 24 h or AEA (10 μM) treatment of BeWo for 48 h were analysed by Giemsa and Höechst staining. These concentrations were selected according to the viability assays of each cellular model. After the treatment, cells plated in 8-well chamber slides were fixed with a 4% paraformaldehyde solution, stained with Giemsa and analysed under light microscopy. For Höechst staining, cells were exposed to 0. 5 mg/ml Höechst 33342 for 20 min and examined under a fluorescence microscope (Eclipse E400, Nikon, Japan) equipped with an excitation filter with maximum transmission at 360/40 nm. 2.5 Incorporation of 3H-thymidine The impact of AEA in BeWo cells proliferation was evaluated by the quantification of 3H-thymidine incorporation in the absence or presence of AEA (1-25 µM) for 24 and 48 h. 3Hthymidine (0.5 μCi final concentration) was added to each well, 8 h before the end of the incubation time. Cells were frozen/thawed twice, harvested with a semi-automated cell harvester (Skatron Instruments, Lier, Norway). After the addition of scintillation cocktail, the incorporation of 3H-thymidine was quantified in a scintillation counter (LS 6500, Beckman Instruments, Fullerton, CA, USA). 3   2.6 Determination of Caspase-3/7, 8 and 9 activities The activity of caspases was determined by luminescence assays by using caspase-Glo®-3/7, 8 or 9 assays, according to manufacture instructions. Cells were seeded in 96-well white plates and exposed to 15 μM of AEA for 20 h (in case of hCTs) or to 10 μM of AEA for 36 h (in case of BeWo cells). The plates were incubated for 1 h at room temperature and the resultant luminescence was measured in a Microplate Luminometer (BioTek Instruments, Winooski, VT, USA) and presented as in relative light units (RLU). A negative control was performed by coincubation of AEA with a specific caspase inhibitor, Z-VAD-fmk (20 μM) and a positive control was performed with the apoptosis inductor staurosporine (STS; 100 nM) added 12 h before the end of experiment. 2.7 Evaluation of mitochondrial membrane potential (∆ψm) and intracellular reactive oxygen and nitrogen species (ROS/RNS) For the assessment of ∆ψm and ROS/RNS production, BeWo cells were seeded in 96-well black plates and treated with AEA (10 μM) for 36 h or 48 h, respectively. For ∆ψm studies, cells were washed and incubated with DiOC6 100 nM, for 20 min, at 37 ºC, in the dark. For the evaluation of ROS/RNS production, cells were washed and incubated with the fluorescent probe 2,7-Dichlorodihydrofluorescein diacetate (DCDHF-DA), for 1 h, at room temperature. For both assays, the resulting fluorescence was measured in a Microplate Fluorimeter (BioTek Instruments, Vermont, USA) (excitation-485+/- 10 nm; emission 530+/-12, 5 nm). The positive controls for ∆ψm or ROS/RNS production were the mitochondrial depolarizing carbonyl cyanide m-chlorophenylhydrazone (CCCP; 10 μM) or H2O2 (200 μM), respectively. The results were expressed in relative fluorescence units (RFU). 2.8 Western Blot analysis Western Blot was used for the assessment of cleaved PARP-1 in hCTs and Bid/t-Bid and IκBα (Table 1). BeWo cells were seeded in 6 well plates and incubated in the absence or presence of AEA alone or pre-incubated with CB receptors antagonists. After 24 or 48 h, for hCTs and BeWo respectively, cells were lysed in icecold lysis buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, 1% TritonTM X-100). Proteins were resolved in SDS-polyacrylamide gel Table 1. Summary of the electrophoresis conditions. All the antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA), except anti-Bid, which was from Cell Signaling Technology (ZA Leiden, Netherlands)  Protein Molecular Weight (kDa) SDS‐PAGE (%) Positive control Primaryantibody Secondaryantibody source dilution sourcedilution IκB‐α 3510%Helacells extractRabbit1:400Goatanti‐rabbit1:2000 Cleaved PARP‐18510% BeWocells treated withSTS Rabbit1:100Goatanti‐rabbit1:1000 BID/t‐BID22/1515%Helacells extractRabbit1:200Goatanti‐rabbit1:1000 β‐tubulin55 Rabbit1:750Goatanti‐rabbit1:3000 electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking nonspecific binding sites, membranes were incubated with the corresponding primary antibody, overnight, at 4 ºC, and then with peroxidase-conjugated secondary antibody for 1 h, at room temperature. Lastly, a Western BrightTM ECL was added to the membranes and exposed to x-ray film. Membranes were then stripped and reincubated with anti-tubulin antibody for loading control. The signal intensity was quantified by densitometry (BIO-PROFIL Bio-1D2; Vilber Lourmat, Marne-la-Vallée, France) and the results expressed in arbitrary 4   units, after normalization for the corresponding β-tubulin band. 2.9 Statistical analysis Statistical analysis was carried out by Wilcoxon signed-rank, Mann-Whitney or Kruskal-Wallis tests followed by multiple comparisons with Dunn’s test (GraphPad PRISM v. 6.0, GraphPad Software, Inc., San Diego, CA, USA). The results are the mean of at least five independent experiments performed in triplicate. Data were expressed as mean ± SEM and differences were considered to be statistically significant at p<0.05. 3. Results 3.1 AEA effects in primary cultures of human cytotrophoblasts Treatment of hCTs with AEA decreased cell viability, in a concentration dependent manner, for concentrations higher than 15 μM (Figure 1A). Similar effects were observed at 12 and 24 h of treatment. LDH release was also evaluated to study the cytotoxic effects of AEA. Only the highest concentration (25 μM) induced a significantly release of this enzyme (Figure 1B). The impact of AEA in hCTs morphology was investigated by Giemsa and Höechst staining. Cells treated with AEA (15 μM) showed chromatin condensation and fragmentation (Figures 1C-F). In addition, it was observed an increase of 28% in caspase-3/7 activity, after 20 h of treatment (Figure 1G) and an increase in PARP-1 cleavage (Figure 1H). 3.2 AEA effects in cell viability and proliferation of BeWo cells The human cytotrophoblasts spontaneously differentiate in vitro into syncytiotrophoblasts, after 72 h of culture [24], hampering the study of the cytotrophoblast cell population in primary cultures. Thus, BeWo cells were chosen as a model to carry out the investigation of the cellular pathways triggered by AEA. These cells are widely used as a representative model of hCTs since they are able to proliferate and do not significantly differentiate without a stimuli, to allow the study of AEA effects on cytotrophoblast viability. Additionally, they express CB receptors and other members of ECS and respond to cannabinoid stimuli [20, 22]. The exposure of BeWo cells to AEA diminished cell viability, in a concentration and time dependent manner (Figure 2A). In fact, a 24 h treatment with 15 μM AEA significantly decreased cell viability by 26%. After a 48 h treatment, a cell viability loss was observed with 10 μM AEA (about 20%) and a dramatic reduction on cell viability was observed with 25 μM (by 74%). At 72 h of treatment, lower concentrations of AEA (5 μM) affected cell viability and 10 μM induced a massive cell death (around 55%). LDH release was assessed in order to evaluate the involvement of a cytotoxic mechanism in AEA effects on cell viability (Figure 2B). Higher concentrations than 25 or 15 μM significantly increased LDH release, after 24/48 h or 72 h of treatment, respectively. The 3H-thymidine incorporation assay showed that BeWo cells proliferation was also decreased by 23% with 15 or 10 μM AEA, after a 24 or 48 h exposure, respectively (Figure 2C). This effect was more notorious with higher concentrations of AEA. In the following experiments that aim to study the cellular pathways involved in AEAinduced cell death, BeWo cells were treated with 10 μM of AEA for 48 h, since in these conditions AEA decreased cell viability and proliferation without inducing LDH release. To clarify the involvement of CB receptors in the cellular mechanisms behind the AEA-induced decrease in BeWo cells viability, cells were pretreated with selective antagonists of CB1 and CB2, AM251 and AM630, respectively, which per se did not interfere with cell viability (Figure 2D). 5 Figure 1. Anandamide (AEA) effects i n human cytotrophoblast cells (hCTs). (A) MTT assay showed that AEA decrease d hCTs viability after 12 and 24 h o f treatment, in a concentration-dependen t way, for concentrations higher than 15 μM and (B) LDH release was only significantly increased for 25 μM. Giemsa staining revealed that the 24 htreatment with 15 μM of AEA induce d chromatin condensation (arrows) (E), i n comparison to the control (C). Höechs t staining confirmed the presence of nuclei with chromatin condensation (arrows) in AEA-treated hCTs (F), in compariso n with the control (D). (G) Caspase 3/7 activity was increased in cells treate d with AEA for 20 h and (H) there is a n increase of PARP-1 cleavage after a 24 h treatment; STS was used as positive control. (*p < 0.05 vs. Control; Wilcoxon signed-rank test for A and B; Mann-Whitney for G and H). LDHLactate dehydrogenase; MTTMethylthiazolyldiphenyl-tetrazolium bromide; STSStaurosporine. 6 Figure 2. Anandamide (AEA) effects in BeWo cells viability an d proliferation, after 24, 48 or 72 h of exposure. (A) MTT assay revealed a decrease in cell viability, in a concentration an d time -dependent manner (fo r concentrations higher than 5 μM). (B) LDH release was significantly increased for concentrations higher than 20 μM, for 24 and 48 h treatments; and then 15 μM, fo r 72 h treatment. (C) The incorporation of 3H-thymidine was decrease for 10 μM or highe r concentrations of AEA. (D) CB receptors antagonists, AM251 an d AM630 (1 μM), did not interfere with cell viability, after a 48 htreatment. (*p < 0.05 vs. Control; Wilcoxon signed-rank test). MTTMethylthiazolyldiphenyltetrazolium bromide; LDHLactate dehydrogenase.  3.3 AEA effects in BeWo cells morphology The morphological alterations induced by AEA in BeWo cells were observed by Giemsa and Höechst staining. AEA-treated cells (10 μM) presented morphological features that are characteristic of apoptosis like chromatin condensation and fragmentation and the presence of apoptotic bodies (Figure 3). 3.4 Study of the mechanisms of cell death triggered by AEA in BeWo cells As morphological studies indicated the presence of apoptotic features, we evaluated AEA effects on the activity of the effector caspases-3/7 and also the involvement of intrinsic pathway, by the assessment of caspase 9 activity and mitochondrial membrane potential (∆ψm). We found that this eCB induced a 16% increase in caspases-3/7 activity, effect that was partially reversed by the CB2 antagonist (Figure 4A). Moreover, AEA caused an increment of 24% of caspase-9 activity (Figure 4B) and a loss of 16% of mitochondrial membrane potential and the effects were partially reversed by both CB receptors antagonists, AM251 and AM630 (Figure 4C). The effects of AEA in the production of ROS/RNS were also evaluated, using the probe DCDHF-DA. AEA dramatically exacerbated the generation of ROS/RNS, in comparison with control. Both CB receptors antagonists were able to partially reverse this effect (Figure 4D). To explore if the extrinsic pathway was triggered by AEA in BeWo cells, we investigated the effects of this eCB in proteins related to the extrinsic pathway, caspase-8 activity and t-Bid, and also in IκB-α, a regulatory protein that inhibits the NF-κB transcription factor. It was detected an increase of 19% in the activity of caspase-8, through a mechanism involving both CB1 and CB2 receptors (Figure 5A) and an increase in the formation of t-Bid (Figure 5B). Moreover, AEA also increased the expression of IκB-α, suggesting that NFκB is not activated (Figure 5C). AEA effects on t-Bid and IκB-α were attenuated by CB1 and CB2 antagonists, though without statistical significance. 7 99  PART III Discussion and general conclusions 101  In the last years, ECS research has unveiled new players in several physiological and pathophysiological processes, such as apoptosis, inflammation, neuromodulation or reproduction. Endocannabinoids have been described as important modulators in embryo transport and implantation, decidualization and labour [140, 141, 410]. Furthermore, an abnormal endocannabinoid signalling has been associated with an impairment of these reproductive events, and concomitantly, with disorders like infertility and miscarriages [410]. A proper placental development is crucial for the success of pregnancy and involves a balanced proliferation, differentiation and apoptosis of the main foeto-placental cells, the trophoblasts [85]. Since these cellular events are tightly regulated by a complex network of hormones, cytokines and other molecules, it was postulated that the eCBs might also have a role during placentation. Although information about eCBs function during placental development is lacking, the presence of AEA metabolic enzymes, NAPEPLD and FAAH, and CB receptors were already described in human placenta [139, 392, 401]. It was also reported that AEA decreases BeWo cells viability, through a CB2 receptor-dependent mechanism [139]. In contrast, the effects and role of the other major endocannabinoid 2-AG in reproductive events have been less explored, especially during placentation. In this study, 2-AG main biosynthetic and hydrolysing enzymes, DAGL-α and MAGL, respectively, were found to be expressed in primary human cytotrophoblasts and syncytiotrophoblasts and in the cytotrophoblast cell model BeWo cells. The expression of the cation channel TRPV1, an AEA receptor, was also demonstrated. There was no differences between CTs and STs in DAGL-α, MAGL and TRPV1 mRNA levels and protein expression, suggesting that the expression is not depend on cell differentiation stage. Thus, the human trophoblasts are provided with the enzymatic machinery required to regulate the in situ levels of both major eCBs, AEA and 2-AG, indicating that these lipid mediators may have a role in placentation. Moreover, TRPV1 expression in trophoblasts raises questions about the role of the endovanniloid signalling during placental development. Although this receptor was initially described in neuronal cells and identified as a modulator of nociception or temperature sensing [205], it is now known that TRPV1 is expressed in several other cell types and participates in other biological processes, such as apoptosis, muscle contraction or inflammation [211, 212, 221]. Furthermore, TRPV1 was identified in rat placenta, though its function remains unclear [394, 411]. 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