On the existence and function of galanin receptor heteromers in the central nervous system

Galanin receptor (GalR) subtypes 1-3 linked to central galanin neurons may form heteromers with each other and other types of G protein-coupled receptors in the central nervous system (CNS). These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15) to modulate the function of different types of glia-neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR-5-HT1A heteromers likely exist with antagonistic GalR-5-HT1A receptor-receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1-5-HT1A heteromers in cellular models with trans-inhibition of the protomer signaling. A GalR1-GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15) in the CNS. Furthermore, a GalR1-GalR2-5-HT1A heterotrimer is postulated to explain why only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR-5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR-NPYY1 receptor interactions in putative GalR-NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1-GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1-GalR2-NPYY2 heterotrimer. Finally, putative GalR-α2-adrenoreceptor heteromers with antagonistic receptor-receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depression

Evaluation of molecular, cellular and behavioral consequences of serotonin 1A receptor deletion

In 1998, three different research groups simultaneously reported increased anxiety-related behavior in tests of conflict in their serotonin 1a (5-HT1a) receptor knockout (KO) line with male mice being more severely affected by 5-HT1a receptor deletion than female KO. Similarly, in the hippocampus, we observed increased dendritic complexity in the stratum radiatum of CA1 pyramidal neurons in male but not in female 5-HT1a receptor KO mice. These observations prompted us to investigate gender- dependent differences of 5-HT1a receptor deletion in hippocampal-related behavioral tasks. Testing our mice in anxiety-related paradigms, we reproduced the original studies showing increased anxiety- related behavior in male 5-HT1a receptor KO mice when compared to male WT mice, but no difference between female 5-HT1a receptor KO and WT mice. Similarly, male 5-HT1a receptor KO mice were impaired in association of aversive stimuli fear conditioning paradigms. We argue that increased dendritic complexity and increased synaptic strength of CA3-CA1 synapses in the stratum radiatum impaired proper signal propagation attributed to overactivation of CA1 pyramidal neurons leading to impaired fear memory of male 5-HT1a receptor KO mice. Similar mechanisms in the ventral hippocampus are likely to have contributed to gender-dependent differences in anxiety-related behavior in our and the original studies from 1998. In this study, we started to shed light on the 5-HT1a receptor downstream signaling pathways involved in dendritogenesis of pyramidal neurons during early postnatal development. We could show that NR2B-containing NMDA receptor during development acts downstream of 5-HT1a receptor and is responsible for increased amount of branching in male 5-HT1a receptor KO mice. Conversely, protein and NR2B mRNA expression was increased in 5-HT1a receptor KO mice at P15. Although the exact signaling cascade of 5-HT1a receptor regulating NR2B-containing NMDA receptor has not been determined, CaMKII is a potential downstream effector to influence transportation and removal of NR2B-containing NMDA receptors to and from the synapse. In contrast, Erk1/2 likely acts downstream of NR2B-containing NMDA receptors and was shown to be sufficient to regulate dendritic branching. Moreover, increased NR2B-containing NMDA receptor mediated cell death via excitotoxicity during development and is likely to be involved in reduced survival of adult born neurons in the hippocampus of 5-HT1a receptor KO male. The convergence of 5-HT1a receptor signaling onto NR2B-containing NMDA receptor signaling enables estrogen to interfere with its downstream pathway via G-protein coupled estrogen receptor 1 activation resulting in normalization of branching and behavior in female 5-HT1a receptor mice. In conclusion, our data strongly suggests a hormone- regulated mechanism that by converging on NR2B-containing NMDA receptor signaling is able to normalize morphology of pyramidal neurons and behavior of female 5-HT1a receptor KO mice. Our findings provide a possible explanation for gender-dependent differences in the occurrence of mental disorders with 5-HT1a receptor abnormalities as a strong predisposing factor. -- En 1998, trois équipes de recherche ont décrit un comportement de type anxieux dans des tests de conflit pour leur souris transgéniques avec une délétion du gène pour le récepteur 5-HT1a de la sérotonine. De plus, les trois groupes rapportent un phénotype plus sévère pour le comportement anxieux chez les souris transgéniques mâles que femelles. Dans l'hippocampe, la région avec la densité de récepteur 5-HT1a la plus élevée dans le télencéphale, nous avons observé dans le stratum radiatum une complexité accrue des arborisations dendritiques des neurones pyramidaux du secteur CA1 chez les souris transgénique mâles mais pas chez les femelles. Cette observation nous a encouragés à initier cette étude sur les différences en fonction du genre utilisant les tests comportementaux en rapport avec les fonctions de l'hippocampe chez les souris déficientes pour le récepteur 5-HT1a.Testant nos souris avec des paradigmes associés à l'anxiété, nous avons reproduit les données originales montrant que les souris transgéniques mâles ont un phénotype plus sévère que les souris mâles sauvages, mais qu'aucune différence n'est observée entre les femelles sauvages et transgéniques. De même, les souris mâles déficientes pour le récepteur 5-HT1a sont handicapées dans les tests de conditionnement au stress avec des stimuli aversifs. Nous faisons l'hypothèse que l'augmentation de la complexité de l'arborisation dendritique et l'augmentation de la force du signal synaptique entres les régions CA3 et CA1 de l'hippocampe dans le stratum radiatum perturbe la propagation du signal nerveux qui conduit à l'hyperactivation des neurones du secteur CA1. Ceci conduit à une mémoire de stress altérée chez les souris mâles déficientes pour le récepteur 5-HT1a. Un mécanisme similaire dans l'hippocampe ventral contribue probablement aux différences en fonction du genre dans les tests pour le comportement de type anxieux qui ont été rapportés dans les études originales de 1998. Les mesures de protéine et de mRNA ont mis en évidence une augmentation de l'expression du récepteur NMDA contenant la sous- unité NR2B dans les souris déficientes pour le récepteur 5-HT1a à P15. Dans les cultures organotypiques d'hippocampe, nous avons commencé à disséquer les messagers secondaires à l'activation du récepteur 5-HT1a qui sont impliqués dans la régulation de la croissance dendritique des neurones pyramidaux pendant la période postnatale précoce. Nous avons démontré que les récepteurs NR2B sont en aval de l'activation du récepteur 5-HT1a et qu'ils sont impliqués dans l'accroissement du nombre de dendrites chez la souris mâle déficiente pour le récepteur 5-HT1a. Bien que la cascade de signalisation du récepteur 5-HT1a pour réguler les récepteurs NMDA contenant le NR2B ne soit pas établie, CaMKII est identifié comme un effecteur potentiel pour altérer le transport du récepteur NMDA à la synapse. D'autre part, Erk1/2 est probablement un messager en aval du NR2B du récepteur NMDA, et a été documenté comme suffisant pour réguler l'arborisation dendritique. L'augmentation de NR2B à la synapse des souris déficientes pour le récepteur 5-HT1a peut conduire à une augmentation de l'excitotoxicité dans les cellules. Nous avons observé une augmentation chez la souris déficiente pour le récepteur 5-HT1a de la mort cellulaire dans des tranches d'hippocampe stimulées, ce qui peut être en relation avec la réduction de la survie des neurones générés dans l'hippocampe de la souris mâle transgénique adulte par rapport à la souris mâle sauvage. De plus, la convergence de la signalisation du récepteur 5-HT1a sur la signalisation de la sous-unité NR2B du récepteur NMDA permet à l'oestrogène d'interférer avec sa voie de signalisation du récepteur de l'oestrogène couplé à une protéine G (GPER-1), ceci permettant à l'oestrogène de réduire la taille de l'arborisation des neurones pyramidaux de CA1 chez la femelle de la souris déficiente pour le récepteur 5-HT1a. En conclusion, nos observations suggèrent fortement qu'un mécanisme hormonal convergeant sur la voie de signalisation de la sous-unité NR2B du récepteur NMDA permet la normalisation de l'exubérance des dendrites des neurones CA1 de l'hippocampe et du comportement des souris femelles déficientes pour le récepteur 5-HT1a. Ceci donne une explication possible pour la différence en fonction du genre dans l'apparition de troubles mentaux avec les variations du récepteur 5-HT1a comme facteur de prédisposition important.

Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Leukocyte trafficking: a special focus on VAP-1 and CLEVER-1

It is crucial that lymphocytes patrol the body against foreign intruders and that leukocytes invade inflamed tissues to ameliorate the infection or injury. The adhesion molecules in leukocytes and endothelial cells play an essential role in the immune response by directing the traffic of leukocytes. However, the same molecules that guide leukocyte traffic under physiological conditions are also involved in pathological situations, when an overly excessive or harmful inflammatory response leads to tissue destruction and organ dysfunction or tumor growth. Vascular adhesion protein-1 (VAP-1) and Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) are endothelial molecules that participate in the adhesion of leukocytes to the endothelia. This study was designed to elucidate, using different inflammation models, the role of VAP-1 and CLEVER-1 in leukocyte migration to the inflamed tissue, and to evaluate the use of antibodies against these molecules as an anti-adhesive therapy. Also, the role of CLEVER-1 during tumorigenesis was studied. Blocking the function of VAP-1 with antibodies significantly decreased the accumulation of leukocytes in the inflamed tissue. Targeting CLEVER-1 prevented cell migration via lymphatic vessels, as well as leukocyte traffic during inflammation. Following the anti-CLEVER-1 antibody treatment the number of immune regulating leukocytes in tumors was reduced, which led to a decrease in tumor growth. However, the normal immune response towards immunization or bacterial infection was not compromised. Thus, VAP-1 and CLEVER-1 are both potential targets for antiinflammatory therapies for preventing the harmful accumulation of leukocytes in inflamed areas. Targeting CLEVER-1 may also inhibit tumor growth by reducing immunosuppressive leukocytes in tumors

The Structure and Function of αI Domains in Collagen Receptor and Leukocyte Integrins

Integrins are heterodimeric, signaling transmembrane adhesion receptors that connect the intracellular actin microfilaments to the extracellular matrix composed of collagens and other matrix molecules. Bidirectional signaling is mediated via drastic conformational changes in integrins. These changes also occur in the integrin αI domains, which are responsible for ligand binding by collagen receptor and leukocyte specific integrins. Like intact integrins, soluble αI domains exist in the closed, low affinity form and in the open, high affinity form, and so it is possible to use isolated αI domains to study the factors and mechanisms involved in integrin activation/deactivation. Integrins are found in all mammalian tissues and cells, where they play crucial roles in growth, migration, defense mechanisms and apoptosis. Integrins are involved in many human diseases, such as inflammatory, cardiovascular and metastatic diseases, and so plenty of effort has been invested into developing integrin specific drugs. Humans have 24 different integrins, four of which are collagen receptor (α1β1, α2β1, α10β1, α11β1) and five leukocyte specific integrins (αLβ2, αMβ2, αXβ2, αDβ2, αEβ7). These two integrin groups are quite unselective having both primary and secondary ligands. This work presents the first systematic studies performed on these integrin groups to find out how integrin activation affects ligand binding and selectivity. These kinds of studies are important not only for understanding the partially overlapping functions of integrins, but also for drug development. In general, our results indicated that selectivity in ligand recognition is greatly reduced upon integrin activation. Interestingly, in some cases the ligand binding properties of integrins have been shown to be cell type specific. The reason for this is not known, but our observations suggest that cell types with a higher integrin activation state have lower ligand selectivity, and vice versa. Furthermore, we solved the three-dimensional structure for the activated form of the collagen receptor α1I domain. This structure revealed a novel intermediate conformation not previously seen with any other integrin αI domain. This is the first 3D structure for an activated collagen receptor αI domain without ligand. Based on the differences between the open and closed conformation of the αI domain we set structural criteria for a search for effective collagen receptor drugs. By docking a large number of molecules into the closed conformation of the α2I domain we discovered two polyketides, which best fulfilled the set structural criteria, and by cell adhesion studies we showed them to be specific inhibitors of the collagen receptor integrins.

MMP-1/PAR-1 signal transduction axis and its prognostic impact in esophageal squamous cell carcinoma

The matrix metalloprotease-1 (MMP-1)/protease-activated receptor-1 (PAR-1) signal transduction axis plays an important role in tumorigenesis. To explore the expression and prognostic value of MMP-1 and PAR-1 in esophageal squamous cell carcinoma (ESCC), we evaluated the expression of two proteins in resected specimens from 85 patients with ESCC by immunohistochemistry. Sixty-two (72.9%) and 58 (68.2%) tumors were MMP-1- and PAR-1-positive, respectively, while no significant staining was observed in normal esophageal squamous epithelium. MMP-1 and PAR-1 overexpression was significantly associated with tumor node metastasis (TNM) stage and regional lymph node involvement. Patients with MMP-1- and PAR-1-positive tumors, respectively, had poorer disease-free survival (DFS) than those with negative ESCC (P = 0.002 and 0.003, respectively). Univariate analysis showed a significant relationship between TNM stage [hazard ratio (HR) = 2.836, 95% confidence interval (CI) = 1.866-4.308], regional lymph node involvement (HR = 2.955, 95%CI = 1.713-5.068), MMP-1 expression (HR = 2.669, 95%CI = 1.229-6.127), and PAR-1 expression (HR = 1.762, 95%CI = 1.156-2.883) and DFS. Multivariate analysis including the above four parameters identified TNM stage (HR = 2.035, 95%CI = 1.167-3.681), MMP-1 expression (HR = 2.109, 95%CI = 1.293-3.279), and PAR-1 expression (HR = 1.967, 95%CI = 1.256-2.881) as independent and significant prognostic factors for DFS. Our data suggest for the first time that MMP-1 and PAR-1 were both overexpressed in ESCC and are novel predictors of poor patient prognosis after curative resection. The MMP-1/PAR-1 signal transduction axis might be a new therapeutic target for future therapies tailored against ESCC.

Sphingosine-1-phosphate-evoked invasion of follicular thyroid cancer cells : evidence for the involvement of HIF-Iα, MMP2 and MMP9

Sphingolipids are widely expressed molecules, which traditionally were considered to have majorly structural properties. Nowadays, however, they are implicated in a wide range of different biological processes. The bioactive lipid sphingosine 1-phosphate (S1P) has emerged during the past decade as one of the most studied molecules due to its proliferative and pro-migratory abilities both during normal physiology and in the pathology of a subset of different diseases. Migration and invasion of cancer cells require changes in cell behavior and modulation of the tissue microenvironment. Tumor aggressiveness is markedly enhanced by hypoxia, in which hypoxia inducible transcription factors 1-2α (HIF-1-2α) are activated to promote metabolism, proliferation and migration. Invasion requires degradation of the extracellular matrix (ECM) achieved by several degrading and remodeling enzymes. Matrix metalloproteinases (MMPs) are broadly expressed and well accepted as proteolytic enzymes with essential roles both in normal physiology and in pathology. Previously, S1P was shown to strongly evoke migration of follicular ML-1 thyroid cancer cells. The objective of this study was to further investigate and understand the mechanisms behind this regulation. In the first project it was demonstrated that S1P enhances the expression and activity of HIF-1α. S1P enhanced the expression of HIF-1α by increasing its synthesis and stability. The S1P-increased HIF-1α was mediated via S1P3, Gi/0, PI3K, PKCβI, ERK1/2, mTOR and translation factors p70S6K and eIF4E. Finally, it was shown that HIF-1α mediated S1P-induced migration. The ECM is constituted of a complex and coordinated assembly of many types of proteins. In order to be able to invade, cells need to break down the ECM, therefore several key players in this event were investigated in the second project. S1P increased the secretion and activity of MMP2 and MMP9 via S1P-receptor 1 and 3 and that these MMPs participated in the S1P-facilitated invasion of ML-1 cells. In this interplay, calpains and Rac1 were involved, both of which are crucial players in migration and invasion. The prognosis for some types of thyroid cancer is relatively good. However, there are forms of thyroid cancers, for which there are no treatments or the current available treatments are inefficient. Thus, new medical interventions are urgently needed. In the third project the significance of the S1P-receptor modulating drug FTY720, which is currently used for the treatment of multiple sclerosis (MS), was studied. The effect of FTY720 was tested on several thyroid cancer cell lines, and it inhibited the proliferation and invasion of all cancer cell lines tested. In ML-1 cells, FTY720 attenuated invasion by blocking signaling intermediates important for migration and invasion of the cells. Moreover, FTY720 inhibited the proliferation of ML-1 cells by increasing the expression of p21 and p27, hence, inducing cell arrest in G1 phase of the cell cycle. Thus, it can be suggested that FTY720 could be used in the treatment of thyroid cancer.

Clever-1 ja sen ilmeneminen konditionaalisesti poistogeenisissä hiirimalleissa

Proteiinit ovat elimistön perusyksiköitä. Niiden oikeanlainen toiminta ja rakenne ovat välttämättömiä solujen tarkoituksenmukaiselle toiminnalle. CLEVER-1 (common lymphatic endothelial and vascular entohelial receptor-1) on tyypin 2 makrofageissa ja sinusoidaalisessa endoteelissä tavallisesti ilmentyvä proteiini, jonka yhteyttä elimistön normaaliin toimintaan ja moniin eri sairaustiloihin on pyritty selvittämään. Tämän syventävien opintojen kirjallisen työn tarkoituksena on koota kirjallisuuskatsaukseen tämänhetkinen tieto CLEVER-1 -molekyylistä sekä kokeellisessa osiossa tutkia kyseisen molekyylin ilmenemistä eri hiirimalleissa. Kirjallisuuskatsauksen artikkelit valittiin PubMed-tietokannasta. Kokeellisessa osiossa perehdyin CLEVER-1:n ilmenemiseen hiiren maksassa, munuaisessa ja keuhkoissa. Tarkoituksena oli tutkia molekyylin ilmenemistä villityypin hiirissä ja verrata ilmenemistä hiiriin, joissa CLEVER-1 on poistettu vain joko makrofageista tai endoteelisoluista (ns. konditionaalisesti poistogeeniset hiirimallit). Ilmenemistä tutkittiin kaksoisfluoresenssivärjäyksillä. Kirjallisuuskatsauksessa osoitan, miten CLEVER-1 on liitetty moniin eri sairaustiloihin, erityisesti syöpäsairauksiin sekä tulehduksellisiin sairauksiin. Lisäksi pyrin hieman kartoittamaan tulevaisuudennäkymiä CLEVER-1:een liittyvässä tutkimuksessa. Kokeellisessa osiossa sain tuloksia, jotka osoittavat, että konditionaalisesti poistogeeniset hiirimallit eivät toimi aivan odotetulla tavalla. CLEVER-1:tä ei ole kyetty poistamaan täysin halutuista kohdesoluista. Havaitsin kuitenkin joitakin eroja ilmenemisessä poistogeenisten ja villityypin hiirten välillä. Lisäksi CLEVER-1 ilmentyy bronkiolaaristen epiteelisolujen alla kaikkien hiirilinjojen keuhkoissa. Näiden solujen alkuperä on vielä toistaiseksi tuntematon.

Régulation de la lipoprotéine lipase macrophagique et de LOX-1 par des facteurs métaboliques. Implications dans l’athérosclérose associée au diabète de type 2.

Les maladies cardiovasculaires (MCV) sont la principale cause de décès dans les pays occidentaux et constituent la principale complication associée au diabète. La lipoprotéine lipase (LPL) est une enzyme clé du métabolisme des lipides et est responsable de l'hydrolyse des lipoprotéines riches en triglycérides (TG). Plusieurs études ont démontré que la LPL sécrétée par les macrophages dans la paroi artérielle est pro-athérogénique. La dysfonction endothéliale caractérise les stades précoces du processus athérosclérotique. Il a été observé qu’un récepteur nouvellement identifié des lipoprotéines de basse densité oxydées (LDLox), le récepteur de type lectine des LDLox (LOX-1), est fortement exprimé dans les lésions athérosclérotiques humaines et dans l’aorte de rats diabétiques, suggérant un rôle clé de LOX-1 dans la pathogénèse de l’athérosclérose diabétique. Au vu du rôle potentiel de la LPL macrophagique et du LOX-1 dans l’athérosclérose associée au diabète de type 2, nous avons évalué la régulation de ces deux molécules pro-athérogéniques par des facteurs métaboliques et inflammatoires augmentés dans le diabète, soit la leptine, l’acide linoléique (LA) et la protéine C-réactive (CRP). Nos résultats démontrent que : 1) Dans les cellules endothéliales aortiques humaines (HAECs), LA augmente l’expression protéique de LOX-1 de façon temps- et dose-dépendante; 2) La pré-incubation de HAECs avec des antioxydants et des inhibiteurs de la NADPH oxydase, de la protéine kinase C (PKC) et du facteur nucléaire-kappa B (NF-kB), inhibe l’effet stimulant de LA sur l’expression protéique de LOX-1; 3) Dans les HAECs traitées avec LA, on observe une augmentation d’expression des isoformes classiques de la PKC; 4) LA augmente de manière significative l’expression génique de LOX-1 ainsi que la liaison des protéines nucléaires extraites des HAECs à la séquence régulatrice NF-kB présente dans le promoteur du gène de LOX-1; 5) LA augmente, via LOX-1, la captation des LDLox par les cellules endothéliales. Pris dans leur ensemble, ces résultats démontrent que LA augmente l’expression endothéliale de LOX-1 in vitro et appuient le rôle clé de LA dans la dysfonction endothéliale associée au diabète. Au vu de nos études antérieures démontrant qu’une expression accrue de LPL macrophagique chez les patients diabétiques de type 2 et que l’augmentation de facteurs métaboliques dans cette maladie, soit l’homocystéine (Hcys), les acides gras et les produits terminaux de glycation (AGE), accroissent l’expression de la LPL macrophagique, nous avons par la suite déterminé l’effet, in vitro, de deux autres facteurs métaboliques et inflammatoires surexprimés dans le diabète, soit la leptine et la CRP, sur l’expression de la LPL macrophagique. Les concentrations plasmatiques de leptine sont élevées chez les patients diabétiques et sont associées à un accroissement des risques cardiovasculaires. Nous avons démontré que : 1) Dans les macrophages humains, la leptine augmente l’expression de la LPL, tant au niveau génique que protéique; 2) L’effet stimulant de la leptine sur la LPL est aboli par la pré-incubation avec un anticorps dirigé contre les récepteurs à la leptine (Ob-R), des inhibiteurs de la PKC et des antioxydants; 3) La leptine augmente l’expression membranaire des isoformes classiques de la PKC et la diminution de l’expression endogène de la PKC, abolit l’effet de la leptine sur l’expression de la LPL macrophagique; 4) Dans les macrophages murins, la leptine augmente le taux de synthèse de la LPL et augmente la liaison de protéines nucléaires à la séquence protéine activée-1 (AP-1) du promoteur du gène de la LPL. Ces observations supportent la possibilité que la leptine puisse représenter un facteur stimulant de la LPL macrophagique dans le diabète. Finalement, nous avons déterminé, in vitro, l’effet de la CRP sur l’expression de la LPL macrophagique. La CRP est une molécule inflammatoire et un puissant prédicteur d’événements cardiovasculaires. Des concentrations élevées de CRP sérique sont documentées chez les patients diabétiques de type 2. Nous avons démontré que : 1) Dans les macrophages humains, la CRP augmente l’expression de la LPL au niveau génique et protéique et la liaison de la CRP aux récepteurs CD32 est nécessaire pour médier ses effets; 2) La pré-incubation de macrophages humains avec des antioxydants, des inhibiteurs de la PKC et de la protéine kinase mitogénique activée (MAPK), prévient l’induction de la LPL par la CRP; 3) La CRP augmente l’activité de la LPL, la génération intracellulaire d’espèces radicalaires oxygénées (ROS), l’expression d’isoformes classiques de la PKC et la phosphorylation des kinases extracellulaires régulées 1/2 (ERK 1/2); 4) Les macrophages murins traités avec la CRP démontrent une augmentation de la liaison des protéines nucléaires à la séquence AP-1 du promoteur du gène de la LPL. Ces données suggèrent que la LPL puisse représenter un nouveau facteur médiant les effets délétères de la CRP dans la vasculopathie diabétique. Dans l’ensemble nos études démontrent le rôle clé de facteurs métaboliques et inflammatoires dans la régulation vasculaire de la LPL et du LOX-1 dans le diabète. Nos données suggèrent que la LPL et le LOX-1 puissent représenter des contributeurs clé de l’athérogénèse accélérée associée au diabète chez l’humain. Mots-clés : athérosclérose, maladies cardiovasculaires, diabète de type 2, macrophage, LPL, cellules endothéliales, LOX-1, stress oxydatif, leptine, LA, CRP.

Évaluation pré-clinique et clinique de deux nouvelles stratégies dans le traitement des maladies cardiovasculaires : activité anti-thrombotique de l'inhibition du récepteur P2Y1 et activité anti-inflammatoire de l'inhibition du récepteur PAR-1.

Alors que la plaquette a un rôle prépondérant dans le maintient de l'hémostase, son implication dans la formation de la thrombose est également incontestée. Il existe à présent des traitements antiplaquettaires mais ceux-ci présentent quelques failles. Le but de ma maîtrise a donc été de caractériser de nouvelles voies d'inhibition de l'activité plaquettaire. Les deux études qui ont été réalisées concernent l'inhibition de récepteurs couplés aux protéines G, soit le récepteur à l'ADP P2Y1 et le récepteur à la thrombine PAR-1. Dans un premier temps, l'étude du récepteur P2Y1 suggère que l'inhibition de celui-ci seul ou en combinaison avec l'inhibition du récepteur P2Y12 présente un potentiel thérapeutique chez des patients coronariens stables. Dans un deuxième temps, l'étude d'un nouvel antiplaquettaire qui est présentement en phase III de son développement clinique, soit le SCH 530348, a été effectuée. Étant un antagoniste du récepteur PAR-1, le SCH 530348 a des effets qui se répercutent à la fois chez la plaquette et les leucocytes qui possèdent eux aussi ce récepteur. Cette deuxième étude suggère que complémentairement à son effet chez la plaquette, cet inhibiteur diminue les marqueurs de l'inflammation systémique.

Variations in the human cannabinoid receptor (CNR1) gene modulate striatal responses to happy faces.

Happy facial expressions are innate social rewards and evoke a response in the striatum, a region known for its role in reward processing in rats, primates and humans. The cannabinoid receptor 1 (CNR1) is the best-characterized molecule of the endocannabinoid system, involved in processing rewards. We hypothesized that genetic variation in human CNR1 gene would predict differences in the striatal response to happy faces. In a 3T functional magnetic resonance imaging (fMRI) scanning study on 19 Caucasian volunteers, we report that four single nucleotide polymorphisms (SNPs) in the CNR1 locus modulate differential striatal response to happy but not to disgust faces. This suggests a role for the variations of the CNR1 gene in underlying social reward responsivity. Future studies should aim to replicate this finding with a balanced design in a larger sample, but these preliminary results suggest neural responsivity to emotional and socially rewarding stimuli varies as a function of CNR1 genotype. This has implications for medical conditions involving hypo-responsivity to emotional and social stimuli, such as autism.

Variation in the human Cannabinoid Receptor (CNR1) gene modulates gaze duration for happy faces

BACKGROUND: Humans from an early age look longer at preferred stimuli, and also typically look longer at facial expressions of emotion, particularly happy faces. Atypical gaze patterns towards social stimuli are common in Autism Spectrum Conditions (ASC). However, it is unknown if gaze fixation patterns have any genetic basis. In this study, we tested if variations in the cannabinoid receptor 1 (CNR1) gene are associated with gaze duration towards happy faces. This gene was selected because CNR1 is a key component of the endocannabinoid system, involved in processing reward, and in our previous fMRI study we found variations in CNR1 modulates the striatal response to happy (but not disgust) faces. The striatum is involved in guiding gaze to rewarding aspects of a visual scene. We aimed to validate and extend this result in another sample using a different technique (gaze tracking). METHODS: 30 volunteers (13 males, 17 females) from the general population observed dynamic emotion expressions on a screen while their eye movements were recorded. They were genotyped for the identical four SNPs in the CNR1 gene tested in our earlier fMRI study. RESULTS: Two SNPs (rs806377 and rs806380) were associated with differential gaze duration for happy (but not disgust) faces. Importantly, the allelic groups associated with greater striatal response to happy faces in the fMRI study were associated with longer gaze duration for happy faces. CONCLUSIONS: These results suggest CNR1 variations modulate striatal function that underlies the perception of signals of social reward such as happy faces. This suggests CNR1 is a key element in the molecular architecture of perception of certain basic emotions. This may have implications for understanding neurodevelopmental conditions marked by atypical eye contact and facial emotion processing, such as ASC.

Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system

BACKGROUND: Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract. METHODS: We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility. KEY RESULTS: The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to approximately 50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit. CONCLUSIONS & INFERENCES: G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.

Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice

Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cepsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCepsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cbeta, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin-stimulated increases in [Ca2+]i and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCepsilon and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCepsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

TNF-α influences the lateral dynamics of TNF receptor I in living cells

In mammalian cells, inflammation is mainly mediated by the binding of tumor necrosis factor alpha to tumor necrosis factor receptor 1. In this study, we investigated lateral dynamics of TNF-R1 before and after ligand binding using high-density single-particle tracking in combination with photoactivated localization microscopy. Our single-molecule data indicates the presence of tumor necrosis factor receptor 1 with different mobilities in the plasma membrane, suggesting different molecular organizations. Cholesterol depletion led to a decrease of slow receptor species and a strong increase in the average diffusion coefficient. Moreover, as a consequence of tumor necrosis factor-alpha treatment, the mean diffusion coefficient moderately increased while its distribution narrowed. Based on our observation, we propose a refined mechanism on the structural arrangement and activation of tumor necrosis factor receptor 1 in the plasma membrane.